CA2522166C - Lambda integrase mutein for use in recombination - Google Patents

Abstract

Provided is a lambda integrase mutein for use in recombination. This mutein finds utility, for example in application to artificial chromosomes, including ACes, that have been engineered to contain available sites for site- specific, recombination-directed integration of DNA of interest as provided herein. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome.

Description

51205-56(D) LAMBDA INTEGRASE MUTEIN FOR USE IN RECOMBINATION
This application is a divisional application of Canadian patent application serial no. 2,441,937 entitled "Chromosome-Based Platforms" which is based on PCT

application no. PCT/US2002/017452 which entered the Canadian National phase on September 17, 2003 and has an effective filing date of May 30, 2002.

FIELD OF INVENTION

Provided is a lambda integrase mutein for use in recombination. This mutein finds utility, for example in application to artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest as provided herein. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome.

=205-56(S) BACKGROUND

Artificial chromosomes A variety of artificial chromosomes for use in plants and animals, particularly higher plants and animals are available. In particular, U.S. Patent Nos. 6,025,155 and 6,077,697 provide heterochromatic artificial chromosomes designated therein as satellite artificial chromosomes (SATACs) and now designated artificial chromosome expression systems (ACes). These chromosomes are prepared by introducing heterologous DNA into a selected plant or animal cell under conditions that result in integration into a region of the chromosome that leads to an amplification event resulting in production of a dicentric chromosome.

05-56(S) =

Subsequent treatment and growth of cells with dicentric chromosomes, including further amplifications, ultimately results in the artificial chromosomes provided therein. In order to introduce a desired heterologous gene (or a plurality of heterologous genes) into the artificial chromosome, the process is repeated introducing the desired heterologous genes and nucleic acids in the initial targeting step. This process is time consuming and tedious.
Hence, more tractable and efficient methods for introducing heterologous nucleic acid molecules into artificial chromosomes, particularly ACes, are needed.

Therefore, it is an object herein to provide engineered artificial chromosomes that permit tractable, efficient and rational engineering of artificial chromosomes.
SUMMARY OF THE INVENTION
Provided herein are artificial chromosomes that permit tractable, efficient and rational engineering thereof. In particular, the artificial chromosomes provided herein contain one or a plurality of loci (sites) for site-specific, recombination-directed integration of DNA. Thus, provided herein are platform artificial chromosome expression systems ("platform ACes") containing single or multiple site-specific, recombination sites.
The artificial chromosomes and ACes artificial chromosomes include plant and animal chromosomes. Any recombinase system that effects site-specific recombination is contemplated for use herein.
in one embodiment, chromosomes, including platform ACes, are provided that contain one or more lambda att sites designed for recombination-directed integration in the presence of lambda integrase, and that are mutated so that they do not require additional factors.
Methods for preparing such chromosomes, vectors for use in the methods, and uses of the resulting chromosomes are also provided.
Platform ACes containing the recombination site(s) and methods for introducing heterologous nucleic acid into such sites and vectors therefor, are provided.
Also provided herein is a bacteriophage lambda (A) integrase site-specific recombination system.
Methods using recombinase mediated recombination target gene expression vectors and/or genes for insertion thereof into platform chromosomes and the resulting chromosomes are provided.
Combinations and kits containing the combinations of vectors encoding a recombinase and integrase and primers for introduction of the site recognized thereby are also provided. The kits optionally include instructions for performing site-directed integration or preparation of ACes containing such sites.
Also provided herein are mammalian and plant cells comprising the artificial chromosomes and ACes.described herein. The cells can be nuclear donor cells, stem cells, such as a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell or an embryonic stem cell.
Also provided is a lamba-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-integrase3. Also provided are transgenic animals and methods for producing a transgenic animal, comprising introducing a ACes into an embryonic cell, such as a stem cell or embryo. The ACes can comprise heterologous nucleic acid that encodes a therapeutic product. The transgenic animal can be a fish, insect, reptile, amphibians, arachnid or mammal. In certain embodiments, the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer.
The platform ACes, including plant and animal ACes, such as MACs, provided herein can be introduced into cells, such as, but-not limited to, animal cells, including mammalian cells, and into plant cells.
Hence plant cells that contain. platform MACs, animal cells that contain platform PACs and other combinations of cells and platform ACes are provided.

-5a-In one aspect, the invention provides a lambda-integraseR mutein comprising an arginine at a position which corresponds to position 174 of the lambda-integraseR mutein as set forth in SEQ ID NO: 37.

In another aspect, the invention provides a lambda-integraseR mutein, wherein the lambda-integraseR
mutein comprises SEQ ID NO: 37.

DESCRIPTION OF FIGURES

FIGURE 1 provides a diagram depicting creation of an exemplary ACes artificial chromosome prepared using methods detailed in U.S. Patent Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183. In this exemplified embodiment, the nucleic acid is targeted to an acrocentric chromosome in an animal or plant, and the = = , heterologous nucleic acid includes a sequence-specific recombination site and marker genes.
FIGURE 2 provides a map of pWEPuro9K, which is a targeting vector derived from the vector pWE15 (GenBank Accession # X65279;
SEQ ID No. 31). Plasmid pWE15 was modified by replacing the Sa/l (Klenow filled)/Smal neomycin resistance encoding fragment with the Pvull/BamHl (Klenow filled) puromycin resistance-encoding fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc., Palo Alto, CA;
GenBank Accession no. U07648; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb Not! fragment from the plasmid pFK1 61 (see Example 1, see, also Csonka et al. (2000) Journal of Cell Science 113:3207-32161; and SEQ ID NO: 118), containing a portion of the mouse rDNA region, was cloned into the Notl site of pWEPuro resulting in plasmid pWEPuro9K.
FIGURE 3 depicts construction of an ACes platform chromosome with a single recombination site, such as IoxP sites or an attP or attB site.
This platform ACes chromosome is an exemplary artificial chromosome with a single recombination site.
FIGURE 4 provides a map of plasmid pSV40-193attPsensePur.
FIGURE 5 depicts a method for formation of a chromosome platform with multiple recombination integration sites, such as attP sites.
FIGURE 6 sets forth the sequences of the core region of attP, attB, attL and attR (SEQ ID Nos. 33-36).
FIGURE 7 depicts insertional recombination of a vector encoding a marker gene, DsRed and an attB site with an artificial chromosome containing an attP site.
FIGURE 8 provides a map of plasmid pCXLamintR (SEQ ID NO:
112), which includes the Lambda integrase (E174R)-encoding nucleic acid.

51205-56(S) =

FIGURE 9 diagrammatically summarizes the platform technology;
marker 1 permits selection of the artificial chromosomes containing the integration site; marker 2, which is promoterless in the target gene expression vector, permits selection of recombinants. Upon recombination with the platform marker 2 is expressed under the control of a promoter resident on the platform.
FIGURE 10 provides the vector map for the plasmid p 18attBZED-5'6XHS4eGFP (SEQ ID NO: 116).
FIGURE 11 provides the vector map for the plasmid p 18attBZEO-3'6XHS4eGFP (SEQ ID NO: 115).
FIGURE 12 provides the vector map for the plasmid p 18attBZEO-(6XHS4) 2eGFP (SEQ ID NO: 110).
FIGURES 13 AND 14 depict the integration of a PCR product by site-specific recombination as set forth in Example 8.
FIGURE 15 provides the vector map for the plasmid pPAC rDNAunit as set forth in Example 9.A.
DETAILED DESCRIPTION OF THE INVENTION
A. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
Where reference is.made to a URL or other such indentifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found, by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

As used herein, nucleic acid refers to single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. Also included in the term "nucleic acid" are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. When referring to probes or primers, optionally labeled, with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique and of low copy number (typically less than 5, preferably less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides long.
As used herein, DNA is meant to include all types and sizes of DNA
molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.
As used herein, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified-nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.
As used herein, heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations and/or in amounts in a genome or cell that differ from that in which it occurs in nature. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not =

normally produced by the cell in which it is expressed. Any DNA or RNA
that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA. Heterologous DNA and RNA may also encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
Examples of heterologous DNA include, but are not limited to, DNA
that encodes a gene product or gene product(s) of interest, introduced for purposes of modification of the endogenous genes or for production of an encoded protein. For example, a heterologous or foreign gene may be isolated from a different species than that of the host genome, or alternatively, may be isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene. Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers traits including, but not limited to, herbicide, insect, or disease resistance; traits, including, but not limited to, oil quality or carbohydrate composition. Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.
As used herein, operative linkage or operative association, or grammatical variations thereof, of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the . =

promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
Alternatively, consensus ribosome binding sites (see, e.g., Kozak (1991) J. Biol. Chem. 266:19867-19870) can be inserted immediately 5' of the start codon and may enhance expression.
As used herein, a sequence complementary to at least a portion of an RNA, with reference to antisense oligonucleotides, means a sequence having sufficient complementarity to be able to hybridize with the RNA, preferably under moderate or high stringency conditions, forming a stable duplex. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. The longer the hybridizing nucleic acid, the more base mismatches it can contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
As used herein, regulatory molecule refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a polypeptide that is capable of enhancing or inhibiting expression of a gene.
As used herein, recognition sequences are particular sequences of nucleotides that a protein, DNA, or RNA molecule, or combinations thereof, (such as, but not limited to, a restriction endonuclease, a modification methylase and a recombinase) recognizes and binds. For example, a recognition sequence for Cre recombinase (see, e.g., SEQ ID
NO:58) is a 34 base pair sequence containing two 13 base pair inverted =

repeats (serving as the recombinase binding sites) flanking an 8 base pair -core and designated loxP (see, e.g., Sauer (1994) Current Opinion in Biotechnology 5:521-527). Other examples of recognition sequences, include, but are not limited to, attB and attP, attR and attL and others (see, e.g., SEQ ID Nos. 8, 41-56 and 72), that are recognized by the recombinase enzyme Integrase (see, SEQ ID Nos. 37 and 38 for the nucleotide and encoded amino acid sequences of an exemplary lambda phage integrase).
The recombination site designated attB is an approximately 33 base pair sequence containing two 9 base pair core-type lnt binding sites and a 7 base pair overlap region; attP (SEQ ID No. 72) is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins IHF, FIS, and Xis (see, e.g., Landy (1993) Current Opinion in Biotechnology 3:699-7071 see, e.g., SEQ ID Nos. 8 and 72).
As used herein, a recombinase is an enzyme that catalyzes the exchange of DNA segments at specific recombination sites. An integrase herein refers to a recombinase that is a member of the lambda (A) integrase family.
As used herein, recombination proteins include excisive proteins, integrative proteins, enzymes, co-factors and associated proteins that are involved in recombination reactions using one or more recombination sites (see, Landy (1993) Current Opinion in Biotechnology 3:699-707). The recombination proteins used herein can be delivered to a cell via an expression cassette on an appropriate vector, such as a plasmid, and the like. In other embodiments, the recombination proteins can be delivered to a cell in protein form in the same reaction mixture used to deliver the desired nucleic acid, such as a platform ACes, donor target vectors, and the like.

As used herein the expression "lox site" means a sequence of nucleotides at which the gene product of the cre gene, referred to herein as Cre, can catalyze a site-specific recombination event. A LoxP
site is a 34 base pair nucleotide sequence from bacteriophage P1 (see, e.g., Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402).
The LoxP site contains two 13 base pair inverted repeats separated by an 8 base pair spacer region as follows: (SEQ ID NO. 57):
ATAACTTCGTATA ATGTATGC TATACGAAGTTAT
E. co/iDH5AIac and yeast strain BSY23 transformed with plasmid pBS44 carrying two loxP sites connected with a LEU2 gene are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53254 and ATCC 20773, respectively. The lox sites can be isolated from plasmid pBS44 with restriction enzymes EcoRI and Sall, or Xhol and BamHl. In addition, a preselected DNA segment can be inserted into pBS44 at either the Sall or BamHI restriction enzyme sites. Other lox sites include, but are not limited to, LoxB, LoxL, LoxC2 and LoxR sites, which are nucleotide sequences isolated from E. coli (see, e.g., Hoess et a/. (1982) Proc. Nat/. Acad. Sci. U.S.A. 79:3398). Lox sites can also be produced by a variety of synthetic techniques (see, e.g., Ito et al. (1982) Nuc. Acid Res. 10:1755 and Ogilvie et al. (1981) Science 270:270).
As used herein, the expression "cre gene" means a sequence of nucleotides that encodes a gene product that effects site-specific recombination of DNA in eukaryotic cells at lox sites. One cre gene can be isolated from bacteriophage P1 (see, e.g., Abremski et al. (1983) Cell 32:1301-1311). E. coli DH1 and yeast strain BSY90 transformed with plasmid pBS39 carrying a cre gene isolated from bacteriophage P1 and a GAL1 regulatory nucleotide sequence are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53255 =

and ATCC 20772, respectively. The cre gene can be isolated from plasmid pBS39 with restriction enzymes Xhol and Sall.
As used herein, site-specific recombination refers to site-specific recombination that is effected between two specific sites on a single nucleic acid molecule or between two different molecules that requires the presence of an exogenous protein, such as an integrase or recombinase.
For example, Cre-lox site-specific recombination can include the following three events:
a. deletion of a pre-selected DNA segment flanked by lox sites;
b. inversion of the nucleotide sequence of a pre-selected DNA segment flanked by lox sites; and c. reciprocal exchange of DNA segments proximate to lox sites located on different DNA molecules.
This reciprocal exchange of DNA segments can result in an integration event if one or both of the DNA molecules are circular. DNA
segment refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source.
Since the lox site is an asymmetrical nucleotide sequence, two lox sites on the same DNA molecule can have the same or opposite orientations with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox = = ' sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the gene product of the cre gene.
Thus, the Cre-lox system can be used to specifically delete, invert, or insert DNA. The precise event is controlled by the orientation of lox DNA
sequences, in cis the lox sequences direct the Cre recombinase to either delete (lox sequences in direct orientation) or invert (lox sequences in inverted orientation) DNA flanked by the sequences, while in trans the lox sequences can direct a homologous recombination event resulting in the insertion of a recombinant DNA.
As used herein, a chromosome is a nucleic acid molecule, and associated proteins, that is capable of replication and segregation within a cell upon cell division. Typically, a chromosome contains a centromeric region, replication origins, telomeric regions and a region of nucleic acid between the centromeric and telomeric regions.
As used herein, a centromere is any nucleic acid sequence that confers an ability to segregate to daughter cells through cell division. A
centromere may confer stable segregation of a nucleic acid sequence, including an artificial chromosome containing the centromere, through mitotic or meiotic divisions, including through both mitotic and meiotic divisions. A particular centromere is not necessarily derived from the same species in which it is introduced, but has the ability to promote DNA segregation in cells of that species.
As used herein, euchromatin and heterochromatin have their recognized meanings. Euchromatin refers to chromatin that stains diffusely and that typically contains genes, and heterochromatin refers to chromatin that remains unusually condensed and that has been thought to be transcriptionally inactive. Highly repetitive DNA sequences (satellite DNA) are usually located in regions of the heterochromatin surrounding the centromere (pericentric or pericentromeric heterochromatin).
Constitutive heterochromatin refers to heterochromatin that contains the highly repetitive DNA which is constitutively condensed and genetically inactive.
As used herein, an acrocentric chromosome refers to a chromosome with arms of unequal length.
As used herein, endogenous chromosomes refer to genomic chrom-osomes as found in a cell prior to generation or introduction of an artificial chromosome.
As used herein, artificial chromosomes are nucleic acid molecules, typically DNA, that stably replicate and segregate alongside endogenous chromosomes in cells and have the capacity to accommodate and express heterologous genes contained therein. It has the capacity to act as a gene delivery vehicle by accommodating and expressing foreign genes contained therein. A mammalian artificial chromosome (MAC) refers to chromosomes that have an active mammalian centromere(s). Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include centromeres that function in plant, insect and avian cells, respectively. A human artificial chromosome (HAC) refers to chromosomes that include centromeres that function in human cells. For exemplary artificial chromosomes, see, e.g., U.S. Patent Nos. 6,025,155; 6,077,697; 5,288,625; 5,712,134;
5,695,967; 5,869,294; 5,891,691 and 5,721,118 and published International PCT application Nos, WO 97/40183 and WO 98/08964.
Artificial chromosomes include those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Patent Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97140183), minichromosomes that contain a de novo centromere (see, U.S. Patent 005-56(S) Nos. 5,712,134, 5,891,691 and 5,288,625), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA and in vitro assembled artificial chromosomes.
As used herein, the term "satellite DNA-based artificial chromosome (SATAC)" is interchangable with the term "artificial chromosome expression system (ACes)". These artificial chromosomes (ACes) include those that are substantially all neutral non-coding sequences (heterochromatin) except for foreign heterologous, typically gene-encoding nucleic acid, that is interspersed within the heterochromatin for the expression therein (see U.S. Patent Nos.
6,025,155 and 6,077,697 and International PCT application No. WO
97/40183), or that is in a single locus as provided herein. Also included are ACes that may include euchromatin and that result from the process described in U.S. Patent Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183 and outlined herein. The delineating structural feature is the presence of repeating units, that are generally predominantly heterochromatin. The precise structure of the ACes will depend upon the structure of the chromosome in which the initial amplification event occurs; all share the common feature of including a defined pattern of repeating units. Generally ACes have more heterochromatin than euchromatin. Foreign nucleic acid molecules (heterologous genes) contained in these artificial chromosome expression systems can include any nucleic acid whose expression is of interest in a particular host cell. Such foreign nucleic acid molecules, include, but are not limited to, nucleic acid that encodes traceable marker proteins (reporter genes), such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively), other reporter .205-56(S) genes, such as fl-galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance. Other examples of heterologous nucleic acid molecules include, but are not limited to, DNA
that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, DNA that encodes other types of proteins, such as antibodies, and DNA that encodes RNA molecules (such as antisense or siRNA molecules) that are not translated into proteins.

As used herein, an artificial chromosome platform, also referred to herein as a "platform ACes" or "ACes platform", refers to an artificial chromosome that has been engineered to include one or more sites for site-specific, recombination-directed integration. In particular, ACes that are so-engineered are provided. Any sites, including but not limited to any described herein, that are suitable for such integration are contemplated. Plant and animal platform ACes are provided. Among the ACes contemplated herein are those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Patent Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA resulting from an amplification event depicted in the referenced patent and herein. Included among the ACes for' use in generating platforms, are artificial chromosomes that introduce and express heterologous nucleic acids in plants.
These include artificial chromosomes that have a centromere derived from a plant, and, also, artificial chromosomes that have centromeres that may be derived from other organisms but that function in plants.

As used herein a "reporter ACes" refers to an ACes that comprises one or a plurality of reporter constructs, where the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.
As used herein, amplification, with reference to DNA, is a process in which segments of DNA are duplicated to yield two or multiple copies of substantially similar or identical or nearly identical DNA segments that are typically joined as substantially tandem or successive repeats or inverted repeats.
As used herein, amplification-based artificial chromosomes are artificial chromosomes derived from natural or endogenous chromosomes by virtue of an amplification event, such as one initiated by introduction of heterologous nucleic acid into rDNA in a chromosome. As a result of such an event, chromosomes and fragments thereof exhibiting segmented or repeating patterns arise. Artificial chromosomes can be formed from these chromosomes and fragments. Hence, amplification-based artificial chromosomes refer to engineered chromosomes that exhibit an ordered segmentation that is not observed in naturally occurring chromosomes and that distinguishes them from naturally occurring chromosomes. The segmentation, which can be visualized using a variety of chromosome analysis techniques known to those of skill in the art, correlates with the structure of these artificial chromosomes. In addition to containing one or more centromeres, the amplification-based artificial chromosomes, throughout the region or regions of segmentation are predominantly made up of nucleic acid units also referred to as "amplicons", that is (are) repeated in the region and that have a similar gross structure. Repeats of an amplicon tend to be of similar size and share some common nucleic acid sequences. For example, each repeat of an amplicon may contain a replication site involved in amplification of chromosome segments and/or Rectified Sleet (Rule 91) some heterologous nucleic acid that was utilized in the initial production of the artificial chromosome. Typically, the repeating units are substantially similar in nucleic acid composition and may be nearly identical.
The amplification-based artificial chromosomes differ depending on the chromosomal region that has undergone amplification in the process of artificial chromosome formation. The structures of the resulting chromosomes can vary depending upon the initiating event and/or the conditions under which the heterologous nucleic acid is introduced, including modification to the endogenous chromosomes. For example, in some of the artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of heterochromatic DNA.
In other artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of euchromatic DNA or may be made up of similar amounts of heterochromatic and euchromatic DNA.
As used herein an amplicon is a repeated nucleic acid unit. In some of the artificial chromosomes described herein, an amplicon may contain a set of inverted repeats of a megareplicon. A megareplicon represents a higher order replication unit. For example, with reference to some of the predominantly heterochromatic artificial chromosomes, the megareplicon can contain a set of tandem DNA blocks (e.g., -7.5 Mb DNA blocks) each containing satellite DNA flanked by non-satellite DNA
or may be made up of substantially rDNA. Contained within the megareplicon is a primary replication site, referred to as the megareplicator, which may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres. Within the megareplicon there may be smaller (e.g., 50-300 kb) secondary replicons.

=

In artificial chromosomes, such as those provided U.S. Patent Nos.
6,025,155 and 6,077,697 and International PCT application No. WO
97/40183, the megareplicon is defined by two tandem blocks (-7.5 Mb DNA blocks in the chromosomes provided therein). Within each artificial chromosome or among a population thereof, each amplicon has the same gross structure but may contain sequence variations. Such variations will arise as a result of movement of mobile genetic elements, deletions or insertions or mutations that arise, particularly in culture. Such variation does not affect the use of the artificial chromosomes or their overall structure as described herein.
As used herein, amplifiable, when used in reference to a chromosome, particularly the method of generating artificial chromosomes provided herein, refers to a region of a chromosome that is prone to amplification. Amplification typically occurs during replication and other cellular events involving recombination (e.g., DNA repair). Such regions include regions of the chromosome that contain tandem repeats, such as satellite DNA, rDNA, and other such sequences.
As used herein, a dicentric chromosome is a chromosome that contains two centromeres. A multicentric chromosome contains more than two centromeres.
As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments is a replicable chromosome. If one of the chromosomes undergoes amplification of primarily euchromatic DNA to produce a fully functional chromosome that is predominantly (at least more than 50%) euchromatin, it is a minichromosome. The remaining chromosome is a formerly dicentric chromosome. If one of the chromosomes undergoes amplification, whereby heterochromatin (such as, for example, satellite DNA) is amplified and a euchromatic portion (such as, for example, an arm) remains, it is referred to as a sausage chromosome. A chromosome that is substantially all heterochromatin, except for portions of heterologous DNA, is called a predominantly heterochromatic artificial chromosome.
Predominantly heterochromatic artificial chromosomes can be produced from other partially heterochromatic artificial chromosomes by culturing the cell containing such chromosomes under conditions such as. BrdU
treatment that destabilize the chromosome and/or growth under selective conditions so that a predominantly heterochromatic artificial chromosome is produced. For purposes herein, it is understood that the artificial chromosomes may not necessarily be produced in multiple steps, but may appear after the initial introduction of the heterologous DNA. Typically, artificial chromosomes appear after about 5 to about 60, or about 5 to about 55, or about 10 to about 55 or about 25 to about 55 or about 35 to about 55 cell doublings after initiation of artificial chromosome generation, or they may appear after several cycles of growth under selective conditions and BrdU treatment.
As used herein, an artificial chromosome that is predominantly heterochromatic (i.e., containing more heterochromatin than euchromatin, typically more than about 50%, more than about 70%, or more than about 90% heterochromatin) may be produced by introducing nucleic acid molecules into cells, such as, for example, animal or plant cells, and selecting cells that contain a predominantly heterochromatic artificial chromosome. Any nucleic acid may be introduced into cells in such methods of producing the artificial chromosomes. For example, the nucleic acid may contain a selectable marker and/or optionally a sequence that targets nucleic acid to the pericentric, heterochromatic region of a chromosome, such as in the short arm of acrocentric chromosomes and = =

nucleolar organizing regions. Targeting sequences include, but are not limited to, lambda phage DNA and rDNA for production of predominantly heterochromatic artificial chromosomes in eukaryotic cells.
After introducing the nucleic acid into cells, a cell containing a predominantly heterochromatic artificial chromosome is selected. Such cells may be identified using a variety of procedures. For example, repeating units of heterochromatic DNA of these chromosomes may be discerned by G-banding and/or fluorescence in situ hybridization (FISH) techniques. Prior to such analyses, the cells to be analyzed may be enriched with artificial chromosome-containing cells by sorting the cells on the basis of the presence of a selectable marker, such as a reporter protein, or by growing (culturing) the cells under selective conditions. It is also possible, after introduction of nucleic acids into cells, to select cells that have a multicentric, typically dicentric, chromosome, a formerly multicentric (typically dicentric) chromosome and/or various heterochromatic structures, such as a megachromosome and a sausage chromosome, that contain a centromere and are predominantly heterochromatic and to treat them such that desired artificial chromosomes are produced. Cells containing a new chromosome are selected. Conditions for generation of a desired structure include, but are not limited to, further growth under selective conditions, introduction of additional nucleic acid molecules and/or growth under selective conditions and treatment with destabilizing agents, and other such methods (see International PCT application No. WO 97/40183 and U.S. Patent Nos.
6,025,155 and 6,077,697).
As used herein, a "selectable marker" is a nucleic acid segment, generally DNA, that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds and compositions. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be identified, such as phenotypic markers, including /3-galactosidase, red, blue and/or green fluorescent proteins (FPs), and cell surface proteins; (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function;
(6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos. 1-5 above (e.g., antisense oligonucleotides or siRNA molecules for use in RNA interference); (7) nucleic acid segments that bind products that modify a substrate (e.g.
restriction endonucleases); (8) nucleic acid segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional, such as for PCR amplification of subpopulations of molecules; and/or (10) nucleic acid segments, which when absent, directly or indirectly confer sensitivity to particular compounds. Thus, for example, selectable markers include nucleic acids encoding fluorescent proteins, such as green fluorescent proteins, $-galactosidase and other readily detectable proteins, such as chromogenic proteins or proteins capable of being bound by an antibody and FACs sorted. Selectable markers such as these, which are not required for cell survival and/or proliferation in the presence of a selection agent, are also referred to herein as reporter molecules. Other selectable markers, e.g., the neomycin phosphotransf erase gene, provide for isolation and identification of cells containing them by conferring properties on the cells that make them resistant to an agent, e.g., a drug such as an antibiotic, that inhibits proliferation of cells that do not contain the marker.
As another example, interference of gene expression by double stranded RNA has been shown in Caenorhabditis elegans, plants, Drosophila, protozoans and mammals. This method is known as RNA
interference (RNAi) and utilizes short, double-stranded RNA molecules (siRNAs). The siRNAs are generally composed of a 19-22bp double-stranded RNA stem, a loop region and a 1-4 bp overhang on the 3' end.
The reduction of gene expression has been accomplished by direct introduction of the siRNAs into the cell (Harborth J et al., 2001, J Cell Sci 114(pt 24):4557-65) as well as the introduction of DNA encoding and expressing the siRNA molecule. The encoded siRNA molecules are under the regulation of an RNA polymerase III promoter (see, e.g., Yu et al.,.-, 2002, Proc Nat Acad Sci USA 99(9);6047-52; Brummelkamp et al., 2002, Science 296(5567):550-3; Miyagishi et al., 2002, Nat Biotechnol 20(5):497-500; and the like). In certain embodiments, RNAi in mammalian cells may have advantages over other therapeutic methods.
For example, producing siRNA molecules that block viral genetic activities in infected cells may reduce the effects of the virus. Platform ACes provided herein encoding siRNA molecule(s) are an additional utilization of the platform ACes technology. The platform ACes could be engineered to encode one or more siRNA molecules to create gene "knockdowns". In one embodiment, a platform ACes can be engineered to encode both the siRNA molecule and a replacement gene. For example, a mouse model or cell culture system could be generated using a platform ACes that has a knockdown of the endogenous mouse gene, by siRNA, and the human gene homolog expressing in place of the mouse gene. The placement of Rectified Sheet (Rule 91) = =

siRNA encoding sequences under the regulation of a regulatable or inducible promoter would allow one to temporally and/or spatially control the knockdown effect of the corresponding gene.
As used herein, a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Generally reporter genes are readily detectable. Examples of reporter genes include, but are not limited to nucleic acid encoding a fluorescent protein, CAT
(chloramphenicol acetyl transferase) (Alton et aL (1979) Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987) Mol. Cell. Biol.
7:725-737); bacterial luciferase (Engebrecht and Silverman (1984) Proc.
Nat/. Acad. Sci. U.S.A. 81:4154-4158; Baldwin et al. (1984) Biochemistry 23:3663-3667); and alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) J. Mol. AppL Gen.
2:101).
As used herein, growth under selective conditions means growth of a cell under conditions that require expression of a selectable marker for survival.
As used herein, an agent that destabilizes a chromosome is any agent known by those skilled in the art to enhance amplification events, and/or mutations. Such agents, which include BrdU, are well known to those skilled in the art.
In order to generate an artificial chromosome containing a particular heterologous nucleic acid of interest, it is possible to include the nucleic acid in the nucleic acid that is being introduced into cells to initiate production of the artificial chromosome. Thus, for example, a nucleic acid can be introduced into a cell along with nucleic acid encoding a selectable marker and/or a nucleic acid that targets to a heterochromatic region of a chromosome. For introducing a heterologous nucleic acid into =

the cell, it can be included in a fragment that includes a selectable marker or as part of a separate nucleic acid fragment and introduced into the cell with a selectable marker during the process of generating the artificial chromosomes. Alternatively, heterologous nucleic acid can be introduced into an artificial chromosome at a later time after the initial generation of the artificial chromosome.
As used herein, the minichromosome refers to a chromosome derived from a multicentric, typically dicentric, chromosome that contains more euchromatic than heterochromatic DNA. For purposes herein, the minichromosome contains a de novo centromere (e.g., a neocentromere).
In some embodiments, for example, the minichromosome contains a centromere that replicates in animals, e.g., a mammalian centromere or in plants, e.g., a plant centromere.
As used herein, in vitro assembled artificial chromosomes or synthetic chromosomes can be either more euchromatic than heterochromatic or more heterochromatic than euchromatic and are produced by joining essential components of a chromosome in vitro.
These components include at least a centromere, a megareplicator, a telomere and optionally secondary origins of replication.
As used herein, in vitro assembled plant or animal artificial chromosomes are produced by joining essential components (at least the centromere, telomere(s), megareplicator and optional secondary origins of replication) that function in plants or animals. In particular embodiments, the megareplicator contains sequences of rDNA, particularly plant or animal rDNA.
As used herein, a plant is a eukaryotic organism that contains, in addition to a nucleus and mitochondria, chloroplasts capable of carrying out photosynthesis. A plant can be unicellular or multicellular and can contain multiple tissues and/or organs. Plants can reproduce sexually or = .

asexually and can be perennial or annual in growth. Plants can also be terrestrial or aquatic. The term "plant" includes a whole plant, plant cell, plant protoplast, plant calli, plant seed, plant organ, plant tissue, and other parts of a whole plant.
As used herein, stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95%, of the cells retain the chromosome. Stability is measured in the presence of a selective agent. Preferably these chromosomes are also maintained in the absence of a selective agent. Stable chromosomes also retain their structure during cell culturing, suffering no unintended intrachromosomal or interchromosomal rearrangements.
As used herein, de novo with reference to a centromere, refers to generation of an excess centromere in a chromosome as a result of incorporation of a heterologous nucleic acid fragment using the methods herein.
As used herein, BrdU refers to 5-bromodeoxyuridine, which during replication is inserted in place of thymidine. BrdU is used as a mutagen; it also inhibits condensation of metaphase chromosomes during cell division.
As used herein, ribosomal RNA (rRNA) is the specialized RNA that forms part of the structure of a ribosome and participates in the synthesis of proteins. Ribosomal RNA is produced by transcription of genes which, in eukaryotic cells, are present in multiple copies. In human cells, the approximately 250 copies of rRNA genes (i.e., genes which encode rRNA) per haploid genome are spread out in clusters on at least five different chromosomes (chromosomes 13, 14, 15, 21 and 22). In mouse cells, the presence of ribosomal DNA (rDNA, which is DNA containing sequences that encode rRNA) has been verified on at least 11 pairs out of 20 mouse chromosomes (chromosomes 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 19) . .

(see e.g., Rowe et al. (1996) Mamm. Genome 7:886-889 and Johnson et al. (1993) Mamm. Genome 4:49-52). In Arabidopsis thaliana the presence of rDNA has been verified on chromosomes 2 and 4 (18S, 5.8S, and 25S rDNA) and on chromosomes 3,4, and 5 (5S rDNA)(see The Arabidopsis Genome Initiative (2000) Nature 408:796-815). In eukaryotic cells, the multiple copies of the highly conserved rRNA genes are located in a tandemly arranged series of rDNA units, which are generally about 40-45 kb in length and contain a transcribed region and a nontranscribed region known as spacer (i.e., intergenic spacer) DNA
which can vary in length and sequence. In the human and mouse, these tandem arrays of rDNA units are located adjacent to the pericentric satellite DNA sequences (heterochromatin). The regions of these chromosomes in which the rDNA is located are referred to as nucleolar organizing regions (NOR) which loop into the nucleolus, the site of ribosome production within the cell nucleus.
As used herein, a megachromosome refers to a chromosome that, except for introduced heterologous DNA, is substantially composed of heterochromatin. Megachromosomes are made up of an array of repeated amplicons that contain two inverted megareplicons bordered by introduced heterologous DNA (see, e.g., Figure 3 of U.S. Patent No.
6,077,697 for a schematic drawing of a megachromosome). For purposes herein, a megachromosome is about 50 to 400 Mb, generally about 250-400 Mb. Shorter variants are also referred to as truncated megachromosomes (about 90 to 120 or 150 Mb), dwarf megachromosomes (-150-200 Mb), and a micro-megachromosome (-50-90 Mb, typically 50-60 Mb). For purposes herein, the term megachromosome refers to the overall repeated structure based on an array of repeated chromosomal segments (amplicons) that contain two inverted megareplicons bordered by any inserted heterologous DNA. The size will be specified.

As used herein, gene therapy involves the transfer or insertion of nucleic acid molecules into certain cells, which are also referred to as target cells, to produce specific products that are involved in preventing, curing, correcting, controlling or modulating diseases, disorders and deleterious conditions. The nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced. Alternatively, the nucleic acid may in some manner mediate expression of DNA that encodes a therapeutic product. This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. Expression of the nucleic acid by the target cells within an organism afflicted with a disease or disorder thereby provides for modulation of the disease or disorder. The nucleic acid encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
For use in gene therapy, cells can be transfected in vitro, followed by introduction of the transfected cells into an organism. This is often referred to as ex vivo gene therapy. Alternatively, the cells can be transfected directly in vivo within an organism.

As used herein, therapeutic agents include, but are not limited to, growth factors, antibodies, cytokines, such as tumor necrosis factors and interleukins, and cytotoxic agents and other agents disclosed herein and = .

known to those of skill in the art. Such agents include, but are not limited to, tumor necrosis factor, a-interferon, fl-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin- I (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), pro-coagulants such as tissue factor and tissue factor variants, pro-apoptotic agents such FAS-ligand, fibroblast growth factors (FGF), nerve growth factor and other growth factors.
As used herein, a therapeutically effective product is a product that is encoded by heterologous DNA that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease.
As used herein, transgenic plants and animals refer to plants and animals in which heterologous or foreign nucleic acid is expressed or in which the expression of a gene naturally present in the plant or animal has been altered by virtue of introduction of heterologous or foreign nucleic acid.
As used herein, IRES (internal ribosome entry site; see, e.g., SEQ
ID No. 27 and nucleotides 2736-3308 SEQ ID No. 28) refers to a region of a nucleic acid molecule, such as an mRNA molecule, that allows internal ribosome entry sufficient to initiate translation, which initiation can be detected in an assay for cap-independent translation (see, e.g., U.S. Patent No. 6,171,821). The presence of an IRES within an mRNA
molecule allows cap-independent translation of a linked protein-encoding sequence that otherwise would not be translated.

. .

Internal ribosome entry site (IRES) elements were first identified in picornaviruses, which elements are considered the paradigm for cap-independent translation. The 5' UTRs of all picornaviruses are long and mediate translational initiation by directly recruiting and binding ribosomes, thereby circumventing the initial cap-binding step. IRES
elements are frequently found in viral mRNA, they are rare in non-viral mRNA. Among non-viral mRNA molecules that contain functional IRES
elements in their respective 5' UTRs are those encoding immunoglobulin heavy chain binding protein (BiP) (Macejak et at (1991) Nature 353:90-94); Drosophila Antennapedia (Oh et at (1992) Genes Dev, 6:1643-1653); D. Ultrabithorax (Ye et at (1997) Mol. Cell Biol.
17:1714-21); fibroblast growth factor 2 (Vagner et at (1995) Mol. Cell Biol. 15:35-44); initiation factor eIF4G (Gan et at (1998) J. BioL Chem.
273:5006-5012); proto-oncogene c-myc (Nanbru et at (1995) J. Biot.
Chem. 272:32061-32066; Stoneley (1998) Oncogene 16:423-428);
IRESH; from the 5'UTR of NRF1 gene (Oumard et al. (2000) Mot and Cell BioL, 20(8):2755-2759); and vascular endothelial growth factor (VEGF) (Stein et at (1998) Mot Cell BioL 18:3112-9).
As used herein, a promoter, with respect to a region of DNA, refers to a sequence of DNA that contains a sequence of bases that signals RNA
polymerase to associate with the DNA and initiate transcription of RNA
(such as pol II for mRNA) from a template strand of the DNA. A promoter thus generally regulates transcription of DNA into mRNA. A particular promoter provided herein is the Ferritin heavy chain promoter (excluding the Iron Response Element, located in the 5'UTR), which was joined to the 37bp Fer-1 enhancer element. This promoter is set forth as SEQ ID
NO:128. The endogenous Fer-1 enhancer element is located upstream of the Fer-1 promoter (e.g., a Fer-1 oligo was cloned proximal to the core promoter).

As used herein, isolated, substantially pure nucleic acid, such as, for example, DNA, refers to nucleic acid fragments purified according to standard techniques employed by those skilled in the art, such as that found in Sambrook eta/. ((2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd edition).
As used herein, expression refers to the transcription and/or translation of nucleic acid. For example, expression can be the transcription of a gene that may be transcribed into an RNA molecule, such as a messenger RNA (mRNA) molecule. Expression may further include translation of an RNA molecule and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA. With respect to an antisense construct, expression may refer to the transcription of the antisense DNA.
As used herein, vector or plasmid refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors and plasmids are well within the level of skill of the art.
As used herein, transformation/transfection refers to the process by which nucleic acid is introduced into cells. The terms transfection and transformation refer to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, =05-56(S) sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722; Potrykus et a!. (1985) Mol. Gen. Genet. 199:169-177; Reich et al. (1986) Biotechnology 4:1001-1004; Klein et al. (1987) Nature 327:70-73; U.S. Patent No.
6,143,949; Paszkowski et a!. (1989) in Cell Culture and Somatic Cell Genetics of P/ants, Vol. 6, Molecular Biology of Plant Nuclear- Genes, eds.
Schell, J and Vasil, L.K. Academic Publishers, San Diego, California, p.
52-68; and Frame et al. (1994) Plant J. 6:941-948), direct uptake using calcium phosphate (CaP04; see,e.g., Wigler et al. (1979) Proc. Natl.
Acad. Sci. U.S.A. 76:1373-1376), polyethylene glycol (PEG)-mediated DNA uptake, lipofection (see, e.g., Strauss (1996) Meth. Mol. Biol.
54:307-327), microcell fusion (see, EXAMPLES, see, also Lambert (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5907-5911; U.S. Patent No. 5,396,767, Sawford et a!. (1987) Somatic Cell Mol. Genet. 73:279-284; Dhar et a!.
(1984) Somatic Cell MoL Genet. 70:547-559; and McNeill-Killary at al.
(1995) Meth. Enzymol. 254:133-152), lipid-mediated carrier systems (see, e.g., Teifel et a!. (1995) Biotechniques 19:79-80; Albrecht et al.
(1996) Ann. Hematol. 72:73-79; Holmen et a!. (1995) In Vitro Cell Dev.
Biol. Anim. 31:347-351; Remy et a!. (1994) Bioconjug. Chem. 5:647-654; Le Bolch et al. (1995) Tetrahedron Lett. 36:6681-6684; Loeffler at al. (1993) Meth. Enzymol. 217:599-618) or other suitable method.
Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell.

As used herein, "delivery," which is used interchangeably with "transfection," refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.
As used herein, injected refers to the microinjection, such as by use of a small syringe, needle, or pipette, for injection of nucleic acid into a cell.
As used herein, substantially homologous DNA refers to DNA that includes a sequence of nucleotides that is sufficiently similar to another such sequence to form stable hybrids, with each other or a reference sequence, under specified conditions.
It is well known to those of skill in this art that nucleic acid fragments with different sequences may, under the same conditions, hybridize detectably to the same "target" nucleic acid. Two nucleic acid fragments hybridize detectably, under stringent conditions over a sufficiently long hybridization period, because one fragment contains a segment of at least about 10, 14 or 16 or more nucleotides in a sequence that is complementary (or nearly complementary) to a substantially contiguous sequence of at least one segment in the other nucleic acid fragment. If the time during which hybridization is allowed to occur is held constant, at a value during which, under preselected stringency conditions, two nucleic acid fragments with complementary base-pairing segments hybridize detectably to each other, departures from exact complementarity can be introduced into the base-pairing segments, and base-pairing will nonetheless occur to an extent sufficient to make hybridization detectable. As the departure from complementarity between the base-pairing segments of two nucleic acids becomes larger, and as =

conditions of the hybridization become more stringent, the probability decreases that the two segments will hybridize detectably to each other.
Two single-stranded nucleic acid segments have "substantially the same sequence", if (a) both form a base-paired duplex with the same segment, and (b) the melting temperatures of the two duplexes in a solution of 0.5 X SSPE differ by less than 1 OoC. If the segments being compared have the same number of bases, then to have "substantially the same sequence", they will typically differ in their sequences at fewer than 1 base in 10. Methods for determining melting temperatures of nucleic acid duplexes are well known (see, e.g., Meinkoth et a/. (1984) Anal. Biochem. 138:267-284 and references cited therein).
As used herein, a nucleic acid probe is a DNA or RNA fragment that includes a sufficient number of nucleotides to specifically hybridize to DNA or RNA that includes complementary or substantially complementary sequences of nucleotides. A probe may contain any number of nucleotides, from as few as about 10 and as many as hundreds of thousands of nucleotides. The conditions and protocols for such hybridization reactions are well known to those of skill in the art as are the effects of probe size, temperature, degree of mismatch, salt concentration and other parameters on the hybridization reaction. For example, the lower the temperature and higher the salt concentration at which the hybridization reaction is carried out, the greater the degree of mismatch that may be present in the hybrid molecules.
To be used as a hybridization probe, the nucleic acid is generally rendered detectable by labeling it with a detectable moiety or label, such as 32P, 3H and '4C, or by other means, including chemical labeling, such as by nick-translation in the presence of deoxyuridylate biotinylated at the 5'-position of the uracil moiety. The resulting probe includes the biotinylated uridylate in place of thymidylate residues and can be detected =

(via the biotin moieties) by any of a number of commercially available detection systems based on binding of streptavidin to the biotin. Such commercially available detection systems can be obtained, for example, from Enzo Biochemicals, Inc. (New York, NY). Any other label known to those of skill in the art, including non-radioactive labels, may be used as long as it renders the probes sufficiently detectable, which is a function of the sensitivity of the assay, the time available (for culturing cells, extracting DNA, and hybridization assays), the quantity of DNA or RNA
available as a source of the probe, the particular label and the means used to detect the label.
Once sequences with a sufficiently high degree of homology to the probe are identified, they can readily be isolated by standard techniques (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press).
As used herein, conditions under which DNA molecules form stable hybrids are considered substantially homologous, and a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a polypeptide. By the term "substantially homologous" is meant having at least 75%, preferably 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.
The terms "homology" and "identity" are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP
computer program. The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. App!. Math. 2:482 (1981). Briefly, the GAP
program defines similarity as the number of aligned symbols (i.e., = =

nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, NucL Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Preferably the two molecules will hybridize under conditions of high stringency. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
Whether any two nucleic acid molecules have nucleotide sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
"identical" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988).
Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.

In general, sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be = =

calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)).
Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al., J
Molec Biol 215:403 (1990)).
Therefore, as used herein, the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide.
For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.

As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference polypeptides.
Identity at a level of, 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.
As used herein: stringency of hybridization in determining percentage mismatch encompass the following conditions or equivalent conditions thereto:
1) high stringency: 0.1 x SSPE or SSC, 0.1 % SDS, 65 C
2) medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 50 C
3) low stringency: 1.0 x SSPE or SSC, 0.1 % SDS, 50 C
or any combination of salt and temperature and.. other reagents that result in selection of the same degree of mismatch or matching. Equivalent conditions refer to conditions that select for substantially the same percentage of mismatch in the resulting hybrids. Additions of ingredients, such as formamide, Ficoll, and Denhardt's solution affect parameters such as the temperature under which the hybridization should be conducted and the rate of.the reaction. Thus, hybridization in 5 X SSC, in 20%
formamide at 42 C is substantially the same as the conditions recited above hybridization under conditions of low stringency. The recipes for SSPE, SSC and Denhardt's and the preparation of deionized formamide are described, for example, in Sambrook et at. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 8;
see, Sambrook or al., vol. 3, p. B.13, see, also, numerous catalogs that describe commonly used laboratory solutions. It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. As used herein, all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.
As used herein, conservative amino acid substitutions, such as those set forth in Table 1, are those that do not eliminate biological activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. co., p.224). Conservative amino acid substitutions are made, for example, in accordance with those set forth in TABLE 1 as follows:

Original residue Conservative substitution Ala (A) Gly; Ser, Abu Arg (R) Lys, orn Asn (N) Gin; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gin Ile (I) Leu; Val; Met; Nle; Nva Leu (L) Ile; Val; Met; Nle; Nva Lys (K) Arg; Gin; Glu Met IM) Leu; Tyr; Ile; NLe Val Ornithine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met; Nle; Nva = =

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.
As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
As used herein, a probe or primer based on a nucleotide sequence includes at least 10, 14, 16, 30 or 100 contiguous nucleotides from the reference nucleic acid molecule.

As used herein, recombinant production by using recombinant DNA
methods refers to the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced.

The terms substantially identical or similar varies with the context as understood by those skilled in the relevant art and generally means at least 40, 60, 80, 90, 95 or 98%.
As used herein, substantially identical to a product means sufficiently similar so that the property is sufficiently unchanged so that the substantially identical product can be used in place of the product.
As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. The vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector =

refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, protein-binding-sequence refers to a protein or peptide sequence that is capable of specific binding to other protein or peptide sequences generally, to a set of protein or peptide sequences or to a particular protein or peptide sequence.
As used herein, a composition refers to any mixture of two or more ingredients. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a combination refers to any association between two or more items.
As used herein, fluid refers to any composition that can flow.
Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
As used herein, a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell.
As used herein, the term "subject" refers to animals, plants, insects, and birds and other phyla, genera and species into which nucleic acid molecules may be introduced. Included are higher organisms, such as mammals, fish, insects and birds, including humans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, hamsters, cats, dogs, horses, chicken and others.

= =

As used herein, flow cytometry refers to processes that use a laser based instrument capable of analyzing and sorting out cells and or chromosomes based on size and fluorescence.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB
Commission on Biochemical Nomenclature (see, (1972) Biochem.

11:942-944).
B. Recombination systems Site-specific recombination systems typically contain three elements: a pair of DNA sequences (the site-specific recombination sequences) and a specific enzyme (the site-specific recombinase). The site-specific recombinase catalyzes a recombination reaction between two site-specific recombination sequences.
A number of different site-specific recombinase systems are available and/or known to those of skill in the art, including, but not limited to: the Cre/lox recombination system using CRE recombinase (see, e.g., SEQ ID Nos. 58 and 59) from the Escherichia coli phage P1 (see, e.g., Sauer (1993) Methods in Enzymology 225:890-900; Sauer et al.
(1990) The New Biologist 2:441-449), Sauer (1994) Current Opinion in Biotechnology 5:521-527; Odell et al. (1990) Mo/ Gen Genet. 223:369-378; Lasko et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:6232-6236;
U.S. Patent No. 5,658,772), the FLP/FRT system of yeast using the FLP
recombinase (see, SEQ ID Nos. 60 and 61) from the 2,1 episome of Saccharomyces cerevisiae (Cox (1983) Proc. Natl. Acad. Sci. U.S.A.
80:4223; Falco et a/. (1982) Ce/l 29:573-584; Golic et aL (1989) Ce//59:499-509; U.S. Patent No. 5,744,336), the resolvases, including Gin recombinase of phage Mu (Maeser et al. (1991) Mol Gen Genet.
230:170-176; Klippel, A. et al (1993) EMBO J. 12:1047-1057; see, e.g., =

SEQ ID Nos. 64-67), Cin, Hin, ad Tn3; the Pin recombinase of E. coli (see, e.g., SEQ ID Nos. 68 and 69; Enomoto et al. (1983) J Bacteriol.
6:663-668), the R/RS system of the pSR1 plasmid of Zygosaccharomyces rouxii (Araki et al. (1992) J. Mol. Biol. 225:25-37; Matsuzaki et al. (1990) J. Bacteriol. 172: 610-618) and site-specific recombinases from Kluyveromyces drosophilarium (Chen et al. (1986) Nucleic Acids Res.
314:4471-4481) and Kluyveromyces waltii (Chen et al. (1992) J. Gen.
Microbio/. 138:337-345). Other systems are known to those of skill in the art (Stark et al. Trends Genet. 8:432-439; Utatsu et al. (1987) J.
Bacteriol. 169:5537-5545; see, also, U.S. Patent No. 6,171,861).
Members of the highly related family of site-specific recombinases, the resolvase family, such as yd, Tn3 resolvase, Hin, Gin, and Cin are also available. Members of this family of recombinases are typically constrained to intramolecular reactions (e.g., inversions and excisions) and can require host-encoded factors. Mutants have been isolated that relieve some of the requirements for host factors (Maeser et at. (1991) Mol. Gen. Genet. 230:170-176), as well as some of the constraints of intramolecular recombination (see, U.S. Patent No. 6,171,861).
The bacteriophage P1 Cre/lox and the yeast FLP/FRT systems are particularly useful systems for site-specific integration, inversion _or excision of heterologous nucleic acid into, and out of, chromosomes, particularly ACes as provided herein. In these systems a recombinase (Cre or FLP) interacts specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences. The sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT).
The FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells (U.S. Patent No. 5,744,386), and, thus, can be used for producing plant artificial chromosome platforms. In = S

general, short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites. The system catalyzes intra- and intermolecular reactions, and, thus, can be used for DNA excision and integration reactions. The recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation.
The site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.
In the Cre-lox system, discovered in bacteriophage P1, recombination between loxP sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Patent No. 5,658,772). This system can be used to insert, invert or excise nucleic acid located between two lox sites.
Cre can be expressed from a vector. Since the lox site is an asymmetrical nucleotide sequence, lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA
segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA
molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region.

Any site-specific recombinase system known to those of skill in the art is contemplated for use herein. It is contemplated that one or a plurality of sites that direct the recombination by the recombinase are introduced into an artificial chromosome to produce platform ACes. The resulting platform ACes are introduced into cells with nucleic acid encoding the cognate recombinase, typically on a vector, and nucleic acid encoding heterologous nucleic acid of interest linked to the appropriate recombination site for insertion into the platform ACes. The recombinase-encoding-nucleic acid may be introduced into the cells on the same vector, or a different vector, encoding the heterologous nucleic acid.
An E. coil phage lambda integrase system for ACes platform engineering and for artificial chromosome engineering is provided (Lorbach et a/. (2000) J. Mo% Bio/ 296:1175-1181). The phage lambda integrase (Landy, A. (1989) Annu. Rev. Biochem. 58:913-94) is adapted herein and the cognate an sites are provided. Chromosomes, including ACes, engineered to contain one or a plurality of att sites are provided, as are vectors encoding a mutant integrase that functions in the absence other factors. Methods using the modified chromosomes and vectors for introduction of heterologous nucleic acid are also provided.
For purposes herein, one or more of the sites (e.g., a single site or a pair of sites) required for recombination are introduced into an artificial chromosome, such as an ACes chromosome. The enzyme for catalyzing site-directed recombination is introduced with the DNA of interest, or separately, or is engineered onto the artificial chromosome under the control of a regulatable promoter.
As described herein, artificial chromosome platforms containing one or multiple recombination sites are provided. The methods and resulting products are exemplified with the lambda phage Att/Int system, but similar methods may be used for production of ACes platforms with other recombination systems.
The Att/Int system and vectors provided herein are not only intended for engineering ACes platforms, but may be used to engineer an Att/Int system into any chromosome. Introduction of att sites into a chromosome will permit engineering of natural chromosomes, such as by permitting targeted integration genes or regulatory regions, and by controlled excision of selected regions. For example, genes encoding a particular trait may be added to a chromosome, such as plant chromosome engineered to contain one or plurality of att sites. Such chromosomes may be used for screening DNA to identify genes. Large pieces of DNA can be introduced into cells and the cells screened phenotypically to select those having the desired trait.
C. Platforms Provided herein are platform artificial chromosomes (platform ACes) containing single or multiple site-specific recombination sites.
Chromosome-based platform technology permits efficient and tractable engineering and subsequent expression of multiple gene targets. Methods are provided that use DNA vectors and fragments to create platform artificial chromosomes, including animal, particularly mammalian, artificial chromosomes, and plant artificial chromosomes. The artificial chromosomes contain either single or multiple sequence-specific recombination sites suitable for the placement of target gene expression vectors onto the platform chromosome. The engineered chromosome-based platform ACes technology is applicable for methods, including cellular and transgenic protein production, transgenic plant and animal production and gene therapy. The platform ACes are also useful for producing a library of ACes comprising random portions of a given genome (e.g., a mammalian, plant or prokaryotic genome) for genomic =05-56(S) =

screening; as well as a library of cells comprising different and/or mutually exclusive ACes therein.
Exemplary of artificial chromosome platforms are those based on ACes. ACes artificial chromosomes are non-viral, self-replicating nucleic acid molecules that function as a natural chromosome, having all the elements required for normal chromosomal replication and maintenance within the cell nucleus. ACes artificial chromosomes do not rely on integration into the genome of the cell to be effective, and they are not limited by DNA carrying capacity and as such the therapeutic gene(s) of interest, including regulatory sequences, can be engineered into the ACes.
In addition, ACes are stable in vitro and in vivo and can provide predictable long-term gene expression. Once engineered and delivered to the appropriate cell or embryo, ACes work independently alongside host chromosomes, for ACes that are predominantly heterochromatin producing only the products (proteins) from the genes it carries. As provided herein ACes are modified by introduction of recombination site(s) to provide a platform for ready introduction of heterologous nucleic acid.
The ACes platforms can be used for production of transgenic animals and plants; as vectors for genetic therapy; for use as protein production systems; for animal models to identify and target new therapeutics; in cell culture for the development and production of therapeutic proteins; and for a variety of other applications.
1. Generation of artificial chromosomes Artificial chromosomes may be generated by any method known to those of skill in the art. Of particular interest herein are the ACes artificial chromosomes, which contain a repeated unit. Methods for production of ACes are described in detail in U.S. Patent Nos. 6,025,155 and 6,077,697.

=

Generation of de novo ACes.
ACes can be generated by cotransfecting exogenous DNA-such as a mammary tissue specific DNA cassette including the gene sequences for a therapeutic protein, with a rDNA fragment and a drug resistance marker gene into the desired eukaryotic cell, such as plant or animal cells, such as murine cells in vitro. DNA with a selectable or detectable marker is introduced, and can be allowed to integrate randomly into pericentric heterochromatin or can be targeted to pericentric heterochromatin, such as that in rDNA gene arrays that reside on acrocentric chromosomes, such as the short arms of acrocentric chromosomes. This integration event activates the "megareplicator" sequence and amplifies the pericentric heterochromatin and the exogenous DNA, and duplicates a centromere. Ensuing breakage of this "dicentric" chromosome can result in the production of daughter cells that contain the substantially-original chromosome and the new artificial chromosome. The resulting ACes contain all the essential elements needed for stability and replication in dividing cells-centromere, origins of replications, and telomeres. ACes have been produced that express marker genes (lacZ, green fluorescent protein, neomycin-resistance, puromycin-resistance, hygromycin-resistance) and genes of interest. Isolated ACes, for example, have been successfully transferred intact to rodent, human, and bovine cells by electroporation, sonoporation, microinjection, and transfection with lipids and dendrimers.
To render the creation of ACes with desired genes more tractable and efficient, "platform" ACes (platform-ACes) can be produced that contain defined DNA sequences for enzyme-mediated homologous DNA
recombination, such as by Cre or FLP recombinases (Bouhassira et al.
(1996) Blood 88(supplement 1):190a; Bouhassira et aL (1997) Blood, 90:3332-3344; Siebler et al. (1997) Biochemistry: 36:1740-1747;

Siebler et al. (1998) Biochemistry 37: 6229-6234; and Bethke et al.
(1997) Nucl. Acids Res. 25:2828-2834), and as exemplified herein the lambda phage integrase. A lox site contains two 13 bp inverted repeats to which Cre-recombinase binds and an intervening 8 bp core region.
Only pairs of sites having identity in the central 6 bp of the core region are proficient for recombination; sites having non-identical core sequences (heterospecific lox sites) do not efficiently recombine with each other (Hoess et al. (1986) Nucleic Acids Res. 14:2287-2300).
Generating acrocentric chromosomes for plant artificial chromosome formation.
In human and mouse cells de novo formation of a satellite DNA
based artificial chromosome (SATAC, also referred to as ACes) can occur in an acrocentric chromosome where the short arm contains only pericentric heterochromatin, the rDNA array, and telomere sequences.
Plant species may not have any acrocentric chromosomes with the same physical structure described, but "megareplicator" DNA sequences reside in the plant rDNA arrays, also known as the nucleolar organizing regions (NOR). A structure like those seen in acrocentric mammalian chromosomes can be generated using site-specific recombination between appropriate arms of plant chromosomes.
Approach Qin et a!. ((1994) Proc. Natl. Acad. Sci. U.S.A. 91:1706-1710, 1994) describes crossing two Nicotiana tabacum transgenic plants. One plant contains a construct encoding a promoterless hygromycin-resistance gene preceded by a lox site (/ox-hpt), the other plant carries a construct containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre DNA recombinase coding region (35S-lox-cre). The constructs were introduced separately by infecting leaf explants with agrobacterium tumefaciens which carries the kanamycin-resistance gene (Kann). The resultant KanR transgenic plants were crossed. Plants that carried the appropriate DNA recombination event were identified by hygromycin-resistance.

Modification of the above for generation of ACes The KanR cultivars are initially screened, such as by FISH, to identify two sets of candidate transgenic plants. One set has one construct integrated in regions adjacent to the pericentric heterochromatin on the short arm of any chromosome. The second set of candidate plants has the other construct integrated in the NOR region of appropriate chromosomes. To obtain reciprocal translocation both sites must be in the same orientation. Therefore a series of crosses are required, Kann plants generated, and FISH analyses performed to identify the appropriate "acrocentric" plant chromosome for de novo plant ACes formation.
2. Bacteriophage lambda integrase-based site-specific recombination system An integral part of the platform technology includes a site-specific recombination system that allows the placement of selected gene targets or genomic fragments onto the platform chromosomes. Any such system may be used. In particular, a method is provided for insertion of additional DNA fragments into the platform chromosome residing in the cell via sequence-specific recombination using the recombinase activity of the bacteriophage lambda integrase. The lambda integrase system is exemplary of the recombination systems contemplated for ACes. Any known recombination system, including any described herein, particularly any that operates without the need for additional factors or that, by virtue of mutation, does not require additional factors, is contemplated.

As noted the lambda integrase system provided herein can be used with natural chromosomes and artificial chromosomes in addition to ACes. Single or a plurality of recombination sites, which may be the same or different, are introduced into artificial chromosomes to produce artificial chromosome platforms.
3. Creation of bacteriophage lambda integrase site-specific recombination system The lambda phage-encoded integrase (designated Int) is a prototypical member of the integrase family. Int effects integration and excision of the phage in and out of the E. co/i genome via recombination between pairs of attachment sites designated attB/attP and attL/attR.
Each att site contains two inverted 9 base pair core Int binding sites and a 7 base pair overlap region that is identical in wild-type att sites. Each site, except for attB contains additional lnt binding sites. In flanking regions, there are recognition sequences for accessory DNA binding proteins, such as integration host factor (IHF), factor for inversion stimulation (FIS) and the phage encoded excision protein (XIS). Except for attB, Int is a heterobivalent DNA-binding protein and, with assistance from the accessory proteins and negative DNA supercoiling, binds simultaneously to core and arm sites within the same att site.
Int, like Cre and FLP, executes an ordered sequential pair of strand exchanges during integrative and excisive recombination. The natural pairs of target sequences for Int, attB and attP or attL and attR are located on the same or different DNA molecules resulting in intra or intermolecular recombination, respectively. For example, intramolecular recombination occurs between inversely oriented attB and attP, or between attL and attR sequences, respectively, leading to inversion of the intervening DNA segment.

=

Like the recombinase systems, such as Cre and FLP, Int directs site-specific recombination. Unlike the other systems, such Cre and FLP, Int generally requires additional protein factors for integrative and excisive recombination and negative supercoiling for integrative recombination.
Hence, the Int system had not been used in eukaryotic targeting systems.
Mutant Int proteins, designated Int-h (El 74K) and a derivative thereof Int-h/21 8(E 1 74K/E21 8K) do not require accessory proteins to perform intramolecular integrative and excisive recombination in co-transfection assays in human cells (Lorbach et al. (2000) J Mol. Biol.
296:1175-1181); wild-type Int does not catalyze intramolecular recombination in human cells harboring target sites attB and attP.
Hence it had been demonstrated that mutant Int can catalyze factor-independent recombination events in human cells.
There has been no demonstration by others that this system can be used for engineering of eukaryotic genomes or chromosomes. Provided herein are chromosomes, including artificial chromosomes, such as but not limited to ACes that contain att sites (e.g., platform ACes), and the use of such chromosomes for targeted integration of heterologous DNA
into such chromosomes in eukaryotic cells, including animal, such as rodent and human, and plant cells. Mutant lnt provided herein is shown to effect site-directed recombination between sites in artificial chromosomes and vectors containing cognate sites.
An additional component of the chromosome-based platform technology is the site-specific integration of target DNA sequences onto the platform. For this the native bacteriophage lambda integrase has been modified to carry out this sequence specific DNA recombination event in eukaryotic cells. The bacteriophage lambda integrase and its cognate DNA substrate att is a member of the site-specific recombinase family that also includes the bacteriophage P1 Cre/lox system as well as the Saccharomyces cerevisiae 2 micron based FLP/FRT system (see, e.g., Landy (1989) Ann. Rev. Biochem 58:913-949; Hoess et al. (1982) Proc.
Natl. Acad. Sci. U.S.A. 79:3398-3402; Broach et al. (1982) Ce/I 29:227-234).
By combining DNA endonuclease and DNA ligase activity these recombinases recognize and catalyze DNA exchanges between sequences flanking the recognition site. During the integration of lambda genome into the E. coil (lambda recombination) genome, the phage integrase (INT) in association with accessory proteins catalyzes the DNA exchange between the attP site of the phage genome and the attB site of the bacterial genome resulting in the formation of attL and attR sites (Figure 6). The engineered bacteriophage lambda integrase has been produced herein to carry out an intermolecular DNA recombination event between an incoming DNA molecule (primarily on a vector containing the bacterial attB site) and the chromosome-based platform carrying the lambda attP
sequence independent of lambda bacteriophage or bacterial accessory proteins.
In contrast to the bi-directional Cre/lox and FLP/FRT system, the engineered lambda recombination system derived for chromosome-based platform technology is advantageously unidirectional because accessory proteins, which are absent, are required for excision of integrated nucleic acid upon further exposure to the lambda Int recombinase.
4. Creation of platform chromosome containing single or multiple sequence-specific recombination sites a. Multiple sites For the creation of a platform chromosome containing multiple, sequence-specific recombination sites, artificial chromosomes are produced as depicted in Figure 5 and Example 3. As discussed above, artificial chromosomes can be produced using any suitable methodology, including those described in U.S. Patent Nos. 5,288,625; 5,712,134;
5,891,691; 6,025,155. Briefly, to prepare artificial chromosomes containing multiple recombination (e.g., integration) sites, nucleic acid (either in the form a one or more plasmids, such as the plasmid pSV40193attPsensePUR set forth in Example 3) is targeted into an amplifiable region of a chromosome, such as the pericentric region of a chromosome. Among such regions are the rDNA gene loci in acrocentric mammalian chromosomes. Hence, targeting nucleic acid for integration into the rDNA region of mammalian acrocentric chromosomes can include the mouse rDNA fragments (for targeting into rodent cell lines) or large human rDNA regions on BAC/PAC vectors (or subclones thereof in standard vectors) for targeting into human acrocentric chromosomes, such as for human gene therapy applications. The targeting nucleic acid generally includes a detectable or selectable marker, such as antibiotic resistance, such as puromycin and hygromycin, a recombination site (such as attP, attB, attL, attR or the like), and/or human selectable markers as required for gene therapy applications. Cells are grown under conditions that result in amplification and ultimately production of ACes artificial chromosomes having multiple recombination (e.g.,integration) sites therein. ACes having the desired size are selected for further engineering.

b. Creation of platform chromosome containing a single sequence-specific recombination site In this method a mammalian platform artificial chromosome is generated containing a single sequence-specific recombination site. In the Example below, this approach is demonstrated using a puromycin resistance marker for selection and a mouse rDNA fragment for targeting into the rDNA locus on mouse acrocentric chromosomes. Other selection markers and targeting DNA sequences as desired and known to those of =

skill in the art can be used. Additional resistance markers include genes conferring resistance to the antibiotics neomycin, blasticidin, hygromycin and zeocin. For applications, such as gene therapy in which potentially immunogenic responses are to be avoided, host, such as human, derived selectable markers or markers detectable with monoclonal antibodies (MAb) followed by fluorescent activated cell sorting (FACS) can be used.
Examples in this class include, but are not limited to: human nerve growth factor receptor (detection with MAb); truncated human growth factor receptor (detection with MAb); mutant human dihydrofolate reductase (DHFR; detectable using a fluorescent methotrexate substrate); secreted alkaline phosphatase (SEAP; detectable with fluorescent substrate);
thymidylate synthase (TS; confers resistance to fluorodeoxyuridine);
human CAD gene (confers resistance to N-phosphonacetyl-L-aspartate (PALA)).
To construct a platform artificial chromosome with a single site, an ACes artificial chromosome (or other artificial chromosome of interest) can be produced containing a selectable marker. A single sequence specific recombination site is targeted onto ACes via homologous recombination. For this, DNA sequences containing the site-specific recombination sequence are flanked with DNA sequences homologous to a selected sequence in the chromosome. For example, when using a chromosome containing rDNA or satellite DNA, such DNA can be used as homologous sequences to target the site-specific recombination sequence onto the chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the chromosome, the DNA is transfected into cells harboring the chromosome. After transfection and integration of the site-specific cassette, homologous recombination events onto the platform = S

chromosome are subcloned and identified, for example by screening single cell subclones via expression of resistance or a fluorescent marker and PCR analysis. In one embodiment, a platform artificial chromosome, such as a platform ACes, that contains a single copy of the recombination site is selected. Examples 2B and 2D exemplify the process, and Figure 3 provides a diagram depicting one method for the creation of a platform mammalian chromosome containing a single sequence-specific recombination site.
5. Lambda integrase mediated recombination of target gene expression vector onto platform chromosome The third component of the chromosome-based platform technology involves the use of target gene expression vectors carrying, for example, genes for gene therapy, genes for transgenic animal or plant production, and those required for cellular protein production of interest.
Using lambda integrase mediated site-specific recombination, or any other recombinase-mediated site-specific recombination, the target gene expression vectors are introduced onto the selected chromosome platform. The use of target gene expression vector permits use of the de novo generated chromosome-based platforms for a wide range of gene targets. Furthermore, chromosome platforms containing multiple attP
sites provides the opportunity to incorporate multiple gene targets onto a single platform, thereby providing for expression of multiple gene targets, including the expression of cellular and genetic regulatory genes and the expression of all or parts of metabolic pathways. In addition to expressing small target genes, such as cDNA and hybrid cDNA/artificial intron constructs, the chromosome-based platform can be used for engineering and expressing large genomic fragments carrying target genes along with its endogenous genomic promoter sequences. This is of importance, for example, where the therapy requires precise cell specific r =

expression and in instances where expression is best achieved from genomic clones rather than cDNA clones. Figure 9 provides a diagram summarizing one embodiment of the chromosome-based technology.
A feature of the target gene expression vector that is of interest to include is a promoterless marker gene, which as exemplified (see, Figure 9) contains an upstream attB site (marker 2 on Figure 9). The nucleic acid encoding the marker is not expressed unless it is placed downstream from a promoter sequence. Using the recombinase technology provided herein, such as the lambda integrase technology (AINTEi,4R on figure 8) provided herein, site-specific recombination between the attB site on the vector and the promoter-attP site (in the "sense" orientation) on the chromosome-based platform results in the expression of marker 2 on the target gene expression vector, thereby providing a positive selection for the lambda INT mediated site-specific recombination event. Site-specific recombination events on the chromosome-based platform versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR.
Examples of suitable marker 2 genes, include, but are not limited to, genes that confer resistance to toxic compounds or antibiotics, fluorescence activated cell sorting (FACS) sortable cell surface markers and various fluorescent markers. Examples of these genes include, but are not limited to, human L26aR (human homolog of Saccharomyces cerevisiae CYH8 gene), neomycin, puromycin, blasticidin, CD24 (see, e.g., US Patents 5,804,177 and 6,074,836), truncated CD4, truncated low affinity nerve growth factor receptor (LNGFR), truncated LDL receptor, truncated human growth hormone receptor, GFP, RFP, BFP.
The target gene expression vectors contain a gene (target gene) for expression from the chromosome platform. The target gene can be expressed using various constitutive or regulated promoter systems = ~.

across various mammalian species. For the expression of multiple target genes within the same target gene expression vector, the expression of the multiple targets can be coordinately regulated via viral-based or human internal ribosome entry site (IRES) elements (see, e.g., Jackson et al. (1990) Trends Biochem Sci. 15: 477-83; Oumard et al. (2000) Mol.
Cell. Biol. 20: 2755-2759). Furthermore, using IRES type elements linked to a downstream fluorescent marker, e.g., green, red or blue fluorescent proteins (GFP, RFP, BFP) allows for the identification of high expressing clones from the integrated target gene expression vector.
In certain embodiments described herein, the promoterless marker can be transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous' nucleic acid encodes a heterologous protein, and wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein.
In addition, the selectable marker and the heterologous nucleic acid can be transcriptionally linked by the presence of a IRES between them. As set forth herein the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic or fluorescent.
Expression from the target gene expression vector integrated onto the chromosome-based platform can be further enhanced using genomic insulator/boundary elements. The incorporation of insulator sequences into the target gene expression vector helps define boundaries in chromatin structure and thus minimizes influence of chromatin position effects/gene silencing on the expression of the target gene (Bell et al.
(1999) Current Opinion in Genetics and Development 9:191-198; Emery et al. (2000) Proc. Nati. Acad. Sci. U.S.A. 97:9150-9155). Examples of insulator elements that can be included onto target gene expression vector in order to optimize expression include, but are not limited to:

1) chicken ,B-globin HS4 element (Prioleau at al. (1999) EMBO J
18: 4035-4048);
2) matrix attachment regions (MAR; see, e.g., Ramakrishnan et a/. (2000) Mol Cell. Biol. 20:868-877);
3) scaffold attachment regions (SAR; see, e.g., Auten et al.
(1999) Human Gene Therapy 10:1389-1399); and 4) universal chromatin opening elements (UCOE; WO/0005393 and W0/0224930) The copy number of the target gene can be controlled by sequentially adding multiple target gene expression vectors containing the target gene onto multiple integration sites on the chromosome platform.
Likewise, the copy number of the target gene can be controlled within an individual target gene expression vector by the addition of DNA
sequences that promote gene amplification. For example, gene amplification can be induced utilizing the dihydrofolate reductase (DHFR) minigene with subsequent selection with methotrexate (see, e.g., Schimke (1984) Cell 37:705-713) or amplification promoting sequences from the rDNA locus (see, e.g., Wegner eta!. (1989) NucL Acids Res. 17:
9909-9932).
6. Platforms with other recombinase system sites A "double lox" targeting strategy mediated by Cre-recombinase (Bethke at a!. (1997) Nucl. Acids Res. 25:2828-2834) can be used. This strategy employs a pair of heterospecific lox sites-/oxA and loxB, which differ by one nucleotide in the 8 bp spacer region. Both sites are engineered into the artificial chromosome and also onto the targeting DNA
vector. This allows for a direct site-specific insertion of a commercially relevant gene or genes by a Cre-catalyzed double crossover event. In essence a platform ACes is engineered with a hygromycin-resistance gene flanked by the double lox sites generating lox-ACes, which is maintained ..

in the thymidine kinase deficient cell, LMtk(-). The gene of interest, for example, for testing purposes, the green fluorescence protein gene, GFP
and a HSV thymidine kinase gene (tk) marker, are engineered between the appropriate lox sites of the targeting vector. The vector DNA is cotransfected with plasmid pBS185 (Life Technologies) encoding the Cre recombinase gene into mammalian cells maintaining the dual-/ox artificial chromosome. Transient expression of the Cre recombinase catalyzes the site-specific insertion of the gene and the tk-gene onto the artificial chromosome. The transfected cells are grown in HAT medium that selects for only those cells that have integrated and expressed the thymidine kinase gene. The HATR colonies are screened by PCR analyses to identify artificial chromosomes with the desired insertion.
To generate the lox-ACes, Lambda-HygR-/ox DNA is transfected into the LMtk(-) cell line harboring the precursor ACes. Hygromycin-resistant colonies are analyzed by FISH and Southern blotting for the presence of a single copy insert on the ACes.
To demonstrate the gene replacement technology, cell lines containing candidate lox-ACes are cotransfected with pTK-GFP-/ox and pBS185 (encoding the Cre recombinase gene) DNA. After transfection, transient expression of plasmid pBS185 will provide sufficient burst of Cre recombinase activity to catalyze DNA recombination at the lox sites.
Thus, a double crossover event between the ACes target and the exogenous targeting plasmid carrying the /oxA and loxB permits the simple replacement of the hygromycin-resistance gene on the lox-ACes for the tk-GFP cassette from the targeting plasmid, with no integration of vector DNA. Transfected cells are grown in HAT-media to select for tk-expression. Correct targeting will result in the generation of HATR, hygromycin sensitive, and green fluorescent cells. The desired integration event is verified by Southern and PCR analyses. Specific PCR primer sets are used to amplify DNA sequences flanking the individual loxA and /oxB
sites on the lox-ACes before and after homologous recombination.
D. Exemplary applications of the Platform ACes Platform ACes are applicable and tractable for different/optimized cell lines. Those that include a fluorescent marker, for example, can be purified and isolated using fluorescent activated cell sorting (FAGS), and subsequently delivered to a target cell. Those with selectable markers provide for efficient selection and provide a growth advantage. Platform ACes allow multiple payload delivery of donor target vectors via a positive-selection site-specific, recombination system, and they allow for the inclusion of additional genetic factors that improve protein production and protein quality.
The construction and use of the platform ACes as provided for each application may be similarly applied to other applications. Particular descriptions are for exemplification.
1. Cellular Protein Production Platform ACes (CPP ACes) As described herein, ACes can be produced from acrocentric chromosomes in rodent (mouse, hamster) cell lines via megareplicator induced amplification of heterochromatin/rDNA sequences. Such ACes are ideal for cellular protein production as well as other applications described herein and known to those of skill in the art. ACes platforms that contain a plurality of recombination sites are particularly suitable for engineering as cellular protein production systems.
In one embodiment, CPP ACes involve a two-component system:
the platform chromosome containing multiple engineering sites and the donor target vector containing a platform-specific recombination site with designed expression cassettes (see Figure 9).
The platform ACes can be produced from any artificial chromosome, particularly the amplification-based artificial chromosomes.

For exemplification, they are produced from rodent artificial chromosomes produced from acrocentric chromosomes using the technology of U.S.
Patent Nos. 6,077,697, and 6,025,155 and published International PCT
application No. WO 97/40183, in which nucleic acid is targeted to the pericentric heterochromatic, and, particularly into rDNA to initiate the replication event(s). The ACes can be produced directly in the chosen cellular protein production cell lines, such as, but not limited to, CHO
cells, hybridomas, plant cells, plant tissues, plant protoplasts, stem cells and plant calli.
a. Platform Construction In the exemplary embodiment, the initial de novo platform construction requires co-transfecting with excess targeting DNA, such as, rDNA or lambda DNA without an attP region, and an engineered selectable marker. The engineered selectable marker should contain promoter, generally a constitutive promoter, such as human, viral, i.e., adenovirus or SV40 promoter, including the human ferritin heavy chain promoter (SEQ ID NO:128), SV40 and EF1 a promoters, to control expression of a marker gene that provides a selective growth advantage to the cell. An example of such a marker gene is the E. coli hisD gene (encoding histidinol dehydrogenase) which is homologous and analogous to the S. typhimurium hisD a dominant marker selection system for mammalian cells previously described (see, Hartman et a/. (1988) Proc.
Natl. Acad. Sci. U.S.A. 85:8047-8051). Since histidine is an essential amino acid in mammals and a nutritional requirement in cell culture, the E.
coli hisD gene can be used to select for histidine prototrophy in defined media. Furthermore more stringent selection can be placed on the cells by including histinol in the medium. Histidinol is itself permeable and toxic to cells. The hisD provides a means of detoxification.

Placed between the promoter and the marker gene is the bacteriophage lambda attP site to use the bacteriophage lambda integrase dependent site-specific recombination system (described herein). The insertion of an attP site downstream of a promoter element provide forward selection of site-specific recombination events onto the platform ACes .
b. Donor Target Vector Construction A second component of the CPP platform ACes system involves the construction of donor target vectors containing a gene product(s) of interest for the CPP platform ACes. Individual donor target vectors can be designed for each gene product to be expressed thus enabling maximum usage of a de novo constructed platform ACes, so that one or a few CPP platform ACes will be required for many gene targets.
A key feature of the donor vector target is the promoterless marker gene containing an upstream attB site (marker 2 on figure 9). Normally the marker would not be expressed unless it is placed downstream of a promoter sequence. As discussed above, using the lambda integrase technology (AINTE174R on Figure 8 and Figure 9), site-specific recombination between the attB site on the vector and the promoter-attP
site on the CPP platform ACes result in the expression of the donor target vector marker providing positive selection for the site-specific event. Site-specific recombination events on the CPP ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR.
In addition, since the lambda integrase reaction is unidirectional, i.e.
excision reaction is not possible, a number of unique targets can be loaded onto the CPP platform ACes limited only by the number of markers available.

=

Additional features of the donor target vector include gene target expression cassettes flanked by either chromatin insulator regions, matrix attachment regions (MAR) or scaffold attachment regions (SAR). The use of these regions will provide a more "open" chromatin environment for gene expression and help alleviate silencing. An example of such a cassette for expressing a monoclonal antibody is described. For this purpose, a strong constitutive promoter, e.g. chicken fl-actin or RNA Poll, is used to drive the expression of the heavy and light chain open reading frames. The heavy and light chain sequences flank a nonattenuated human IRES (IRESH; from the 5'UTR of NRF1 gene; see Oumard et al., 2000, Mol. and Ce/l Bio/., 20(8):2755-2759) element thereby coordinating transcription of both heavy and light chain sequence. Distal to the light chain open reading frame resides an additional viral encoded IRES (IRESV modified ECMV internal ribosomal entry site (IRES)) element attenuating the expression of the fluorescent marker gene hrGFP from Renilla (Stratagene). By linking the hrGFP with an attenuated IRES, the heavy and light chains along with the hrGFP are monocistronic. Thus, the identification of hrGFP fluorescing cells will provide a means to detect protein producing cells. In addition, high producing cell lines can be identified and isolated by FACS thereby decreasing the time frame in finding high expressers. Functional monoclonal antibody will be confirmed by ELISA.
c. Additional components in cellular protein production platform ACes (CPP Aces) In addition to the aforementioned CPP ACes system, other genetic factors can be included to enhance the yield and quality of the expressed protein. Again to provide maximum flexibility, these additional factors can be inserted onto the CPP platform ACes by AINTE1 74R dependent site-specific recombination. Other factors that could be used with a CPP

Platform ACes include for example, adenovirus E1 a transactivation system which upregulates both cellular and viral promoters (see, e.g., Svensson and Akusjarvi (1984) EMBO 3:789-794; and US patents 5,866,359; 4,775,630 and 4,920,211).
d. Targets for CHO-ACes engineering to enhance cell growth, such as CHO cell growth and protein production/ quality If adding these additional factors onto the CPP ACes is not prudent or desired, the host cell, CHO cells, can be engineered to express these factors (see, below, targets for CHO-ACes engineering to enhance CHO
cell growth and protein production/quality). Additional factors to consider including are addition of insulin or IGF-1 to sustain viabililty;
human sialyltransferases or related factors to produce more human-like glycoproteins; expression of factors to decrease ammonium accumulation during cell growth; expression of factors to inhibit apoptosis; expression of factors to improve protein secretion and protein folding; and expression of factors to permit serum-free transfection and selection.
1) Addition of insulin or IGF-1 to sustain viabililty Stimulatory factors and/or their receptors are expressed to set up an autocrine loop, to improve cell growth, such as CHO cell growth. Two exemplary candidates are insulin and IGF-1 (see, Biotechnol Prog 2000 Sep;16(5):693-7). Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures. Insulin and IGF-1 analog (LongR(3) serve as growth and viability factors for CHO cells.
CHO cells were modified to produce higher levels of essential nutrients and factors. A serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44 cells) was prepared. Chinese hamster ovary cells (DG44 cells), which are normally maintained in 10% serum medium, were gradually weaned to 0.5%
serum medium to increase the probability of successful growth in SF
medium (see, Kim et al. (199) In Vitro Cell Dev Biol Anim 35(4):178-82).
A SF medium (SF-DG44) was formulated by supplementing the basal medium with these components; basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/I) and thymidine (10 mg/1).
Development of a SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.
2) Human sialyltransferases or related factors to produce more human-like glycoproteins CHO cells have been modified by increasing their ability to process protein via addition of complex carbohydrates. This has been achieved by overexpression of relevant processing enzymes, or in some cases, reducing expression of relevant enzymes (see, Bragonzi et aI. (2000) Biochim Biophys Acta 1474(3):273-282; see, also Weikert et al. (1999) Nature biotech. 17:1116-11121; Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95). A CHO cell line expressing alpha 2,6-sialyltransferase was developed for the production of human-like sialylated recombinant glycoproteins. The sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA into the cells. Glycoproteins produced by such CHO cells display alpha2,6-and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins.

As another example for improving the production of human-like sialylated recombinant glycoproteins, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA (see, Ferrari J et a/.
(1998) Biotechnol Bioeng 60(5):589-95). Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40%, wild-type values.
Oligosaccharide biosynthesis pathways in mammalian cells have been engineered for generation of recombinant glycoproteins (see, e.g., Sburlati (1998) Biotechnol Prog 14(2):189-92), which describes a Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins. This cell line was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III) (see, also, Prati et al. (1998) Biotechnol Bioeng 59(4):445-50, which describes antisense strategies for glycosylation engineering of CHO cells).
3) Expression of factors to decrease ammonium accumulation during cell growth Excess ammonium, which is a by-product of CHO cell metabolism can have detrimental effects on cell growth and protein quality (see, Yang et al. (2000) Biotechnol Bioeng 68(4):370-80). To solve this problem .ammonium levels were modified by overexpressing carbamoyl phosphate synthetase I and ornithine transcarbamoylase or glutamine synthetase in CHO cells. Such modification resulted in reduced ammonium levels observed and an increase in the growth rate (see Kim et al. (2000) J
Biotechnol 89(2-3):129-40; and Enosawa et al. (1997) Cell Transplant 6(5):537-40).

4) Expression of factors to improve protein secretion and protein folding =

Overexpression of relevant enzymes can be engineered into the ACes to improve protein secretion and folding.
5) Expression of factors to permit serum-free transfection and selection It is advantageous to have the ability to convert CHO cells in suspension growing in serum free medium to adherence with out having to resort to serum addition. Laminin or fibronectin addition is sufficient to make cells adherent (see, e.g., Zaworski et ai. (1993) Biotechniques 15(5):863-6) so that expressing either of these genes in CHO cells under an inducible promoter should allow for reversible shift to adherence without requiring serum addition.
2. Platform ACes and Gene Therapy The platform ACes provided herein are contemplated for use in mammalian gene therapy, particularly human gene therapy. Human ACes can be derived from human acrocentric chromosomes from human host cells, in which the amplified sequences are heterochromatic and/or human rDNA. Different platform ACes applicable for different tissue cell types are provided. The ACes for gene therapy can contain a single copy of a therapeutic gene inserted into a defined location on platform ACes.
Therapeutic genes include genomic clones, cDNA, hybrid genes and other combinations of sequences. Preferred selectable markers are those from the mammalian host, such as human derived factors so that they are non-immunogenic, non-toxic and allow for efficient selection, such as by FACS and/or drug resistance.
-25 Platform ACes, useful for gene therapy and other applications, as noted herein, can be generated by megareplicator dependent amplification, such as by the methods in U.S. Patent Nos. 6,077,697 and 6,025,155 and published International PCT application No.
WO 97/40183. In one embodiment, human ACes are produced using human rDNA constructs that target rDNA arrays on human acrocentric chromosomes and induce the megareplicator in human cells, particularly in primary cell lines (with sufficient number of doublings to form the ACes) or stem cells (such as hematopoietic stem cells, mesenchymal stem cells, adult stem cells or embryonic stem cells) to avoid the introduction of potentially harmful rearranged DNA sequences present in many transformed cell lines. Megareplicator induced ACes formation can result in multiple copies of targeting DNA/selectable markers in each amplification block on both chromosomal arms of the platform ACes.
In view of the considerations regarding immunogenicity and toxicity, the production of human platform ACes for gene therapy applications employs a two component system analogous to the platform ACes designed for cellular protein production (CPP platform ACes). The system includes a platform chromosome of entirely human DNA origin containing multiple engineering sites and a gene target vector carrying the therapeutic gene of interest.
a. Platform Construction The initial de novo construction of the platform chromosome employs the co-transfection of excess targeting DNA and a selectable marker. In one embodiment, the DNA is targeted to the rDNA arrays on the human acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22). For example, two large human rDNA containing PAC clones 18714 and 18720 and the human PAC clone 558F8 are used for targeting (Genome Research (ML) now Incyte, BACPAC Resources, 747 52nd Street, Oakland CA). The mouse rDNA clone pFK161 (SEQ ID NO: 118), which was used to make the human SATAC from the 94-3 hamster/human hybrid cell line (see, e.g., published International PCT
application No. WO 97140183 and Csonka, et al, Journal of Cell Science 113:3207-32161 and Example 1 for a description of pFK161) can also be used.
For animal applications, selectable markers should be non-immunogenic in the animal, such as a human, and include, but are not limited to: human nerve growth factor receptor (detected with a MAb, such as described in US patent 6,365,373); truncated human growth factor receptor (detected with MAb), mutant human dihydrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine);
human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+ cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2a; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectable by Ara-C).
Since megareplicator induced amplification generates multiple copies of the selectable marker, a second consideration for the selection of the human marker is the resulting dose of the expressed marker after ACes formation. High level of expression of certain markers may be detrimental to the cell and/or result in autoimmunity. One method to decrease the dose of the marker protein is by shortening its half-life, such as via the fusion of the well-conserved human ubiquitin tag (a 76 amino acid sequence) thus leading to increased turnover of the selectable marker. This has been used successfully for a number of reporter Rectifi She t (RAIL Val]

S.. .

systems including DHFR (see, e.g., Stack et al. (2000) Nature Biotechnology 78:1298-1302 and references cited therein).
Using the ubiquitin tagged protein, a human selectable marker system analogous to the CPP ACes described herein is constructed.
Briefly, a tagged selectable marker, such as for example one of those described herein, is cloned downstream of an attP site and expressed from a human promoter. Exemplary promoters contemplated for use herein include, but are not limited to, the human ferritin heavy chain promoter (SEQ ID NO:128); RNA Poll; EF1 a; TR; glyceraldehyde-3-phosphate dehydrogenase core promoter (GAP); a GAP core promoter including a proximal insulin inducible element and the intervening GAP
sequence; phosphofructokinase promoter; and phosphoglycerate kinase promoter. Also contemplated herein is an aldolase A promoter H1 & H2 (representing closely spaced transcriptional start sites) along with the proximal H enhancer. There are 4 promoters (e.g., transcriptional start sites) for this gene, each having different regulatory and tissue activity.
The H (most proximal 2) promoters are ubiquitously expressed off the H
enhancer. This resulting marker can then be co-transfected along with excess human rDNA targeting sequence into the host cells. An important criteria for the selection of the recipient cells is sufficient number of cell doublings for the formation and detection of ACes. Accordingly, the co-transfections should be attempted in human primary cells that can be cultured for long periods of time, such as for example, stem cells (e.g., hematopoietic, mesenchymal, adult or embryonic stem cells), or the like. Additional cell types, include, but are not limited to: single gene transfected cells exhibiting increased life-span; over-expressing c-myc cells, e.g. MSU1.1 (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136); over-expressing telomerase lines, Rectified Sheet (Rule 91) =

such as TERT cells; SV40 large T-antigen transfected lines; tumor cell lines, such as HT1080; and hybrid human cell lines, such as the 94-3 hamster/human hybrid cell line.
b. Gene Target Vector The second component of the GT platform ACes (GT ACes) system involves the use of engineered target vectors carrying the therapeutic gene of interest. These are introduced onto the GT platform ACes via site-specific recombination. As with the CPP ACes, the use of engineered target vectors maximizes the use of the de novo generated GT platform ACes for most gene targets. Furthermore, using lambda integrase technology, GT platform ACes containing multiple attP sites permits the opportunity to incorporate multiple therapeutic targets onto a single platform. This could be of value in cases where a defined therapy requires multiple gene targets, a single therapeutic target requires an additional gene regulatory factor or a GT ACes requires a "kill" switch.
Similar to the CPP ACes, a feature of the gene target vector is the promoterless marker gene containing an upstream attB site (marker 2 on Figure 9). Normally, the marker (in this case, a cell surface antigen that can be sorted by FACS would be ideal) would not be expressed unless it is placed downstream of a promoter sequence. Using the lambda integrase technology (AINTE1411 on figure 9), site-specific recombination between the attB site on the vector and the promoter- attP site on the GT
platform ACes results in the expression of marker#2 on the gene target vector, i.e. positive selection for the site-specific event. Site-specific recombination events on the GT ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR.
For expression of the therapeutic gene, human specific promoters, such as a ferritin heavy chain promoter (SEQ ID N0:128); EF1 a or RNA

Poll, are used'. These promoters are for high level expression of a cDNA
encoded therapeutic protein. In addition to expressing cDNA (or even hybrid cDNA/artificial intron constructs), the GT platform ACes are used for engineering and expressing large genomic fragments carrying therapeutic genes of interest expressed from native promoter sequences.
This is of importance in situations where the therapy requires precise cell specific expression or in instances where expression is best achieved from genomic clones versus cDNA.
3. Selectable markers for use, for example, in Gene Therapy (GT) The following are selectable markers that can be incorporated into human ACes and used for selection.
Dual Resistance to 4-Hydroperoxycyclophosphamide and Methotrexate by Retroviral Transfer of the Human Aldehyde Dehydrogenase Class 1 Gene and a Mutated Dihydrofolate Reductase Gene The genetic transfer of drug resistance to hematopoietic cells is one approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors, a retroviral vector containing a human cytosolic ALDH-1-encoding DNA clone and a human doubly mutated DHFR-encoding clone (Phe22/Ser3l; termed F/S in the description of constructs) to generate increased resistance to CTX and MTX were constructed (Takebe et a/. (2001) Mol Ther 3(1):88-96). This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression. ACes can be similarly constructed.

= i.

Multiple mechanisms of N-phosphonacetyl-L-aspartate resistance in human cell lines: carbamyl-P
synthetase/aspartate transcarbamylase/dihydro-orotase gene amplification is frequent only when chromosome 2 is rearranged Rodent cells resistant to N-phosphonacetyl-L-aspartate (PALA) invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells (Smith et a/. (1997) Proc.
Nat/. Acad. Sci. U.S.A. 94:1816-21). In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD.
These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts. Thus, ACes with multiple copies of the CAD gene would provide PALA resistance.
Retroviral coexpression of thymidylate synthase and dihydrofolate reductase confers fluoropyrimidine and antifolate resistance Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. Fantz et al.
((1998) Biochem Biophys Res Comm 243(7):6-12) have shown that S

retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR
cytotoxic agents. Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold). Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30-to 140-fold). A L22YhDHFR-Y33HhTS chimeric retroviral vector was-also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA
that encodes TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents. ACes can be similarly constructed.

Human CD34+ cells do not express glutathione S-transferases alpha The expression of glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+ cells and bone marrow was studied using RT-PCR and immunoblotting (Czerwinski M, Kiem et al. (1997) Gene Ther 4(3):268-70). The GSTA1 protein conjugates glutathione to the stern cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body. Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow. Similarly, baboon CD34+ cells and dog bone marrow do not express GSTA1. Thus, human = , .

GSTA1 is a chemoprotective selectable marker in human stem cell gene therapy and could be employed in ACes construction.
Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer Pawliuk et al. ((1994) Blood 84(9):2868-2877) have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells.
The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony-forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. These results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker permits rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and provides a tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.
ACes could be similarly constructed.

DeltahGHR, a biosafe cell surface-labeling molecule for analysis and selection of genetically transduced human cells A selectable marker for retroviral transduction and selection of human and murine cells is known (see, Garcia-Ortiz et a/. (2000) Hum Gene Ther 11(2):333-46). The molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering. The engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding. This new marker, has a wide application spectrum, since hGHR
in the human adult is highly expressed only in liver cells, and lower levels have been reported in certain lymphocyte cell populations. The deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans. The differential binding properties of several monoclonal antibodies (MAbs) are used in a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population is recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium. Such a system could be used to identify ACes containing cells.
4. Transgenic models for evaluation of genes and discovery of new traits in plants Of interest is the use of plants and plant cells containing artificial chromosomes for the evaluation of new genetic combinations and discovery of new traits. Artificial chromosomes, by virtue of the fact that they can contain significant amounts of DNA can also therefore encode =

numerous genes and accordingly a multiplicity of traits. It is contemplated here that artificial chromosomes, when formed from one plant species, can be evaluated in a second plant species. The resultant phenotypic changes observed, for example, can indicate the nature of the genes contained within the DNA contained within the artificial chromosome, and hence permit the identification of novel genetic activities. Artificial chromosomes containing euchromatic DNA or partially containing euchromatic DNA can serve as a valuable source of new traits when transferred to an alien plant cell environment. For example, it is contemplated that artificial chromosomes derived from dicot plant species can be introduced into monocot plant species by transferring a dicot artificial chromosome. The dicot artificial chromosome possessing a region of euchromatic DNA containing expressed genes.
The artificial chromosomes can be designed to allow the artificial chromosome to recombine with the naturally occurring plant DNA in such a fashion that a large region of naturally occurring plant DNA becomes incorporated into the artificial chromosome. This allows the artificial chromosome to contain new genetic activities and hence carry novel traits. For example, an artificial chromosome can be introduced into a wild relative of a crop plant under conditions whereby a portion of the DNA present in the chromosomes of the wild relative is transferred to the artificial chromosome. After isolation of the artificial chromosome, this naturally occurring region of DNA from the wild relative, now located on the artificial chromosome can be introduced into the domesticated crop species and the genes encoded within the transferred DNA expressed and evaluated for utility. New traits and gene systems can be discovered in this fashion. The artificial chromosome can be modified to contain sequences that promote homologous recombination within plant cells, or be modified to contain a genetic system that functions as a site-specific recombination system.
Artificial chromosomes modified to recombine with plant DNA offer many advantages for the discovery and evaluation of traits in different plant species. When the artificial chromosome containing DNA from one plant species is introduced into a new plant species, new traits and genes can be introduced. This use of an artificial chromosome allows for the ability to overcome the sexual barrier that prevents transfer of genes from one plant species to another species. Using artificial chromosomes in this fashion allows for many potentially valuable traits to be identified including traits that are typically found in wild species. Other valuable applications for artificial chromosomes include the ability to transfer large regions of DNA from one plant species to another, such as DNA encoding potentially valuable traits such as altered oil, carbohydrate or protein composition, multiple genes encoding enzymes capable of producing valuable plant secondary metabolites, genetic systems encoding valuable agronomic traits such as disease and insect resistance, genes encoding functions that allow association with soil bacterium such as growth promoting bacteria or nitrogen fixing bacteria, or genes encoding traits that confer freezing, drought or other stress tolerances. In this fashion, artificial chromosomes can be used to discover regions of plant DNA that encode valuable traits.
The artificial chromosome can also be designed to allow the transfer and subsequent incorporation of these valuable traits now located on the artificial chromosome into the natural chromosomes of a plant species. In this fashion the artificial chromosomes can be used to transfer large regions of DNA encoding traits normally found in one plant species into another plant species. In this fashion, it is possible to derive a plant cell that no longer needs to carry an artificial chromosome to =

posses the novel trait. Thus, the artificial chromosome would serve as the transfer mechanism to permit the formation of plants with greater degree of genetic diversity.
The design of an artificial chromosome to accomplish the afore-mentioned purposes can include within the artificial chromosome the presence of specific DNA sequences capable of acting as sites for homologous recombination to take place. For example, the DNA
sequence of Arabidopsis is now known. To construct an artificial chromosome capable of recombining with a specific region of Arabidopsis DNA, a sequence of Arabidopsis DNA, normally located near a chromosomal location encoding genes of potential interest can be introduced into an artificial chromosome by methods provided herein. It may be desirable to include a second region of DNA within the artificial chromosome that provides a second flanking sequence to the region encoding genes of potential interest, to promote a double recombination event which would ensure transfer of the entire chromosomal region, encoding genes of potential interest, to the artificial chromosome. The modified artificial chromosome, containing the DNA sequences capable of homologous recombination region, can then be introduced into Arabidopsis cells and the homologous recombination event selected.
It is convenient to include a marker gene to allow for the selection of a homologous recombination event. The marker gene is preferably inactive unless activated by an appropriate homologous recombination event. For example, US 5,272,071, describes a method where an inactive plant gene is activated by a recombination event such that desired homologous recombination events can be easily scored. Similarly, US 5,501,967 describes a method for the selection of homologous recombination events by activation of a silent selection gene first introduced into the plant DNA, the gene being activated by an appropriate homologous recombination event. Both of these methods can be applied to enable a selective process to be included to select for recombination between an artificial chromosome and a plant chromosome. Once the homologous recombination event is detected, the artificial chromosome, once selected, is isolated and introduced into a recipient cell, for example, tobacco, corn, wheat or rice, and the expression of the newly introduced DNA sequences evaluated.
Phenotypic changes in the recipient plant cells containing the artificial chromosome, or in regenerated plants containing the artificial chromosome, allows for the evaluation of the nature of the traits encoded by the Arabidopsis DNA, under conditions naturally found in plant cells, including the naturally occurring arrangement of DNA sequences responsible for the developmental control of the traits in the normal chromosomal environment.
Traits such as durable fungal or bacterial disease resistance, new oil and carbohydrate compositions, valuable secondary metabolites such as phytosterols, flavonoids, efficient nitrogen fixation or mineral utilization, resistance to extremes of drought, heat or cold are all found within different populations of plant species and are often governed by multiple genes. The use of single gene transformation technologies does not permit the evaluation of the multiplicity of genes controlling many valuable traits. Thus, incorporation of these genes into artificial chromosomes allows the rapid evaluation of the utility of these genetic combinations in heterologous plant species.
The large scale order and structure of the artificial chromosome provides a number of unique advantages in screening for new utilities or novel phenotypes within heterologous plant species. The size of new DNA that can be carried by an artificial chromosome can be millions of base pairs of DNA, representing potentially numerous genes that may = =

have novel utility in a heterologous plant cell. The artificial chromosome is a "natural" environment for gene expression, the problems of variable gene expression and silencing seen for genes transferred by random insertion into a genome should not be observed. Similarly, there is no need to engineer the genes for expression, and the genes inserted would not need to be recombinant genes. Thus, one expects the expression from the transferred genes to be temporal and spatial, as observed in the species from where the genes were initially isolated. A valuable feature for these utilities is the ability to isolate the artificial chromosomes and to further isolate, manipulate and introduce into other cells artificial chromosomes carrying unique genetic compositions.
Thus, the use of artificial chromosomes and homologous recombination in plant cells can be used to isolate and identify many valuable crop traits.
In addition to the use of artificial chromosomes for the isolation and testing of large regions of naturally occurring DNA, methods for the use of artificial chromosomes and cloned DNA are also contemplated. Similar to that described above, artificial chromosomes can be used to carry large regions of cloned DNA, including that derived from other plant species.
The ability to incorporate novel DNA elements into an artificial chromosome as it is being formed allows for the development of artificial chromosomes specifically engineered as a platform for testing of new genetic combinations, or "genomic" discoveries for model species such as Arabidopsis. It is known that specific "recombinase" systems can be used in plant cells to excise or re-arrange genes. These same systems can be used to derive new gene combinations contained on an artificial chromosome.
The artificial chromosomes can be engineered as platforms to accept large regions of cloned DNA, such as that contained in Bacterial S

Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs).
It is further contemplated, that as a result of the typical structure of artificial chromosomes containing tandemly repeated DNA blocks, that sequences other than cloned DNA sequence can be introduced by recombination processes. In particular recombination within a predefined region of the tandemly repeated DNA within the artificial chromosome provides a mechanism to "stack" numerous regions of cloned DNA, including large regions of DNA contained within BACs or YACs clones.
Thus, multiple combinations of genes can be introduced onto artificial chromosomes and these combinations tested for functionality. In particular, it is contemplated that multiple YACs or BACs can be stacked onto an artificial chromosomes, the BACs or YACs containing multiple genes of complex pathways or multiple genetic pathways. The BACs or YACs are typically selected based on genetic information available within the public domain, for example from the Arabidopsis Information Management System (http://aims.cps.msu.edu/aims/index.html) or the information related to the plant DNA sequences available from the Institute for Genomic Research (http://www.tigr.org) and other sites known to those skilled in the art. Alternatively, clones can be chosen at random and evaluated for functionality. It is contemplated that combinations providing a desired phenotype can be identified by isolation of the artificial chromosome containing the combination and analyzing the nature of the inserted cloned DNA.
In this regard, it is contemplated that the use of site-specific recombination sequences can have considerable utility in developing artificial chromosomes containing DNA sequences recognized by recombinase enzymes and capable of accepting DNA sequences containing same. The use of site-specific recombination as a means to target an introduced DNA to a specific locus has been demonstrated in =

the art and such methods can be employed. The recombinase systems can also be used to transfer the cloned DNA regions contained within the artificial chromosome to the naturally occurring plant or mammalian chromosomes.
As noted herein, many site-specific recombinases are known and can be identified (Kilby et al. (1993) Trends in Genetics 9:413-418). The three recombinase systems that have been extensively employed include:
an activity identified as R encoded by the pSR1 plasmid of Zygosaccharomyes rouxii, FLP encoded for the 2um circular plasmid from Saccharomyces cerevisiae and Cre-lox from the phage P1.
The integration function of site-specific recombinases is contemplated as a means to assist in the derivation of genetic combinations on artificial chromosomes. In order to accomplish this, it is contemplated that a first step of introducing site-specific recombinase sites into the genome of a plant cell in an essentially random manner is conducted, such that the plant cell has one or more site-specific recombinase recognition sequences on one or more of the plant chromosomes. An artificial chromosome is then introduced into the plant cell, the artificial chromosome engineered to contain a recombinase recognition site (e.g., integration site) capable of being recognized by a site-specific recombinase. Optionally, a gene encoding a recombinase enzyme is also included, preferably under the control of an inducible promoter. Expression of the site-specific recombinase enzyme in the plant cell, either by induction of a inducible recombinase gene, or transient expression of a recombinase sequence, causes a site-specific recombination event to take place, leading to the insertion of a region of the plant chromosomal DNA (containing the recombinase recognition site) into the recombinase recognition site of the artificial chromosome, and forming an artificial chromosome containing plant chromosomal DNA.

The artificial chromosome can be isolated and introduced into a heterologous host, preferably a plant host, and expression of the newly introduced plant chromosomal DNA can be monitored and evaluated for desirable phenotypic changes. Accordingly, carrying out this recombination with a population of plant cells wherein the chromosomally located recombinase recognition site is randomly scattered throughout the chromosomes of the plant, can lead to the formation of a population of artificial chromosomes, each with a different region of plant chromosomal DNA, and each potentially representing a novel genetic combination.
This method requires the precise site-specific insertion of chromosomal DNA into the artificial chromosome. This precision has been demonstrated in the art. For example, Fukushige and Sauer ((1992) Proc. Natl. Acad. Sci. USA, 89:7905-7909) demonstrated that the Cre-/ox homologous recombination system could be successfully employed to introduce DNA into a predefined locus in a chromosome of mammalian cells. In this demonstration a promoter-less antibiotic resistance gene modified to include a lox sequence at the 5' end of the coding region was introduced into CHO cells. Cells were re-transformed by electroporation with a plasmid that contained a promoter with a lox sequence and a transiently expressed Cre recombinase gene. Under the conditions employed, the expression of the Cre enzyme catalyzed the homologous recombination between the lox site in the chromosomally located promoter-less antibiotic resistance gene, and the lox site in the introduced promoter sequence, leading to the formation of a functional antibiotic resistance gene. The authors demonstrated efficient and correct targeting of the introduced sequence, 54 of 56 lines analyzed corresponded to the predicted single copy insertion of the DNA due to Cre catalyzed site-specific homologous recombination between the lox sequences.

Accordingly a lox sequence may be first added to a genome of a plant species capable of being transformed and regenerated to a whole plant to serve as a recombinase target DNA sequence for recombination with an artificial chromosome. The lox sequence may be optimally modified to further contain a selectable marker which is inactive but can be activated by insertion of the lox recombinase recognition sequence into the artificial chromosome.
A promoterless marker gene or selectable marker gene linked to the recombinase recognition sequence, which is first inserted into the chromosomes of a plant cell can be used to engineer a platform chromosome. A promoter is linked to a recombinase recognition site, in an orientation that allows the promoter to control the expression of the marker or selectable marker gene upon recombination within the artificial chromosome. Upon a site-specific recombination event between a recombinase recognition site in a plant chromosome and the recombinase recognition site within.the introduced artificial chromosome, a cell is derived with a recombined artificial chromosome, the artificial chromosome containing an active marker or selectable marker activity that permits the identification and or selection of the cell.
The artificial chromosomes can be transferred to other plant or animal species and the functionality of the new combinations tested. The ability to conduct such an inter-chromosomal transfer of sequences has been demonstrated in the art. For example, the use of the Cre-lox recombinase system to cause a chromosome recombination event between two chromatids of different chromosomes has been shown.
Any number of recombination systems may be employed as described herein, such as, but not limited to, bacterially derived systems such as the att/int system of phage lambda, and the Gin/gix system.

=

More than one recombination system may be employed, including, for example, one recombinase system for the introduction of DNA into an artificial chromosome, and a second recombinase system for the subsequent transfer of the newly introduced DNA contained within an artificial chromosome into the naturally occurring chromosome of a second plant species. The choice of the specific recombination system used will be dependent on the nature of the modification contemplated.
By having the ability to isolate an artificial chromosome, in particular, artificial chromosomes containing plant chromosomal DNA
introduced via site-specific recombination, and re-introduce the chromosome into other mammalian or plant cells, particularly plant cells, these new combinations can be evaluated in different crop species without the need to first isolate and modify the genes, or carry out multiple transformations or gene transfers to achieve the same combination isolation and testing combinations of the genes in plants.
The use of a site-specific recombinase also allows the convenient recovery of the plant chromosomal region into other recombinant DNA
vectors and systems, such as mammalian or insect systems, for manipulation and study.
Also contemplated herein are ACes, cell lines and methods for use in screening a new chromosomal combinations, deletions, truncations with eucaryotic genome that take advantage of the site-specific recombination systems incorporated onto platform ACes provided herein.
For example, provided herein is a cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes. Also provided herein is a method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into a cell line comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell's chromosomal DNA; and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within. Also provided herein is a library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein. The library of cells can be from a different species and/or cell type than the chromosomal nucleic acid within the ACes. Also provided is a method of making one or more cell lines, comprising a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites, b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell's endogenous chromosomal DNA;
c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein;
d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells;
e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines.
These ACes, cell lines and methods utilize the site-specific recombination sites on platform ACes analogous YAC manipulation related to: the methods of generating terminal deletions in normal and artificial chromosomes (e.g., ACes; as described in Vollrath et al., 1988, PNAS, USA, 85:6027-66031; and Pavan et at., PNAS, USA, 87:1300-1304); the methods of generating interstitial deletions in normal and artificial chromosomes (as described in Campbell et al., 1991, PNAS, USA, 888:5744-5748); and the methods of detecting homologous recombination between two ACes (as described in Cellini et at., 1991, Nuc. Acid Res., 19(5):997-1000).
5. Use of plateform ACes in Pharmacogenomic/toxicology applications (development of "Reporter ACes") In addition to the placement of genes onto ACes chromosomes for therapeutic protein production or gene therapy, the platform can be engineered via the IntR lambda integrase to carry reporter-linked constructs (reporter genes) that monitor changes in cellular physiology as measured by the particular reporter gene (or a series of different reporter genes) readout.
The reporter linked constructs are designed to include a gene that can be detected (by for example fluorescence, drug resistance, immunohistochemistry, or transcript production, and the like) with well-known regulatory sequences that would control the expression of the detectable gene. Exemplary regulatory promoter sequences are well-known in the art, A) Reporter ACes for drug pathway screening The ACes can be engineered to carry reporter-linked constructs that indicate a signal is being transduced through one or a number of pathways.
For example, transcriptionally regulated promoters from genes at the end (or any other chosen point) of particular signal transduction pathways could be engineered on the ACes to express the appropriate readout (either by fluorescent protein production or drug resistance) when the pathway is activated (or down-regulated as well). In one embodiment, a number of reporters from different pathways can be placed on an Rectified Sheet (Rule v1) =

ACes chromosome. Cells (and/or whole animals) containing such a Reporter ACes could be exposed to a variety of drugs or compounds and monitored for the effects of the drugs or compounds upon the selected pathway(s) by the reporter gene(s). Thus, drugs or compounds can be classified or identified by particular pathways they excite or down-regulate. Similarly, transcriptional profiles obtained from genomic array experiments can be biologically validated using the reporter ACes provided herein.
B) Reporter ACes for toxic compound testing Environmental or man-made genotoxicants can be tested in cell lines carrying a number of reporter-genes platform ACes linked to promoters that are transcriptionally regulated in response to DNA damage, induced apoptosis or necrosis, and cell-cycle perturbations. Furthermore, new drugs and/or compounds could be tested in a similar manner with the genotoxicant ACes reporter for their cellular/genetic toxicity by such a screen. Likewise, toxic compound testing could be carried out in whole transgenic animals carrying the ACes chromosome that measures genotoxicant exposure ("canary in a coal mine"). Thus, the same or similar type ACes could be used for toxicity testing in either a cell-based or whole animal setting. An example would include ACes that carry reporter-linked genes controlled by various cytochrome P450 profiled promoters and the like.
C) Reporter ACes for individualized pharmacogenomics/drug profiling A common disease may arise via various mechanisms. In many instances there are multiple treatments available for a given disease.
However, the success of a given treatment may depend upon the mechanism by which the disease originated and/or by the genetic background of the patient. In order to establish the most effective treatment for a given patient one could utilize the ACes reporters provided herein. ACes reporters can be used in patient cell samples to determine an individualized drug regimen for the patient. In addition, potential polymorphisms affecting the transcriptional regulation of an individual's particular gene can be assessed by this approach.
D) Reporter ACes for classification of similar patient tumors As with other diseases as described in 5.C) above, cancer cells arise via different mechanisms. Furthermore, as a cancerous cell propagates it may undergo genomic alterations. An ACes reporter transferred to cells of different patients having the same disease, i.e.
similar cancers, could be used to categorize the particular cancer of each patient, thereby facilitating the identification of the most effective therapeutic regimen. Examples would include the validation of array profiling of certain classes of breast cancers. Subsequently, appropriate drug profiling could be carried out as described above.
E) Reporter ACes as a "differentiation" sensor Using the ACes reporter as a "differentiation" sensor in stem cells or other progenitor cells in order to enrich by selection (either FACS based screening, drug selection and/or use of suicide gene) for a particular class of differentiated or undifferentiated cells. For example, in one embodiment, this assay could also be used for compound screening for small molecule modifiers of cell differentiation.
F) Whole animal studies with Reporter ACes Finally, with whole-body fluorescence imaging technology (Yang et al. (2000) PNAS 97:12278) any of the above Reporter ACes methods could be used in conjunction with whole-body imaging to monitor reporter genes within whole animals without sacrificing the animal. This would allow temporal and spatial analysis of expression patterns under a given set of conditions. The conditions tested may include for example, normal =

differentiation of a stem cell, response to drug or compound treatment whether targeted to the diseased tissue or presented systemically, response to genotoxicants, and the like.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

pFK161 Cosmid pFK161 (SEQ ID NO: 118) was obtained from Dr. Gyula Hadlaczky and contains a 9 kb Notl insert derived from a murine rDNA
repeat (see clone 161 described in PCT Application Publication No.
W097/40183 by Hadlaczky et al. for a description of this cosmid). This cosmid, referred to as clone 161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQ ID NO. 26. It was produced by inserting fragments of the megachromosome (see, U.S. Patent No.
6,077,697 and International PCT application No. WO 97/40183). For example, H 1 D3, which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome into plasmid pWE15 (Stratagene, La Jolla, California; SEQ ID No. 31) as follows. Half of a 100,uI low melting point agarose block (mega-plug) containing isolated SATACs was digested with Notl overnight at 37 C.
Plasmid pWE15 was similarly digested with Notl overnight. The mega-plug was then melted and mixed with the digested plasmid, ligation buffer and T4 DNA ligase. Ligation was conducted at 16 C overnight. Bacterial DH5a cells were transformed with the ligation product and transformed cells were plated onto LB/Amp plates. Fifteen to twenty colonies were grown on each plate for a total of 189 colonies. Plasmid DNA was isolated from colonies that survived growth on LB/Amp medium and analyzed by Southern blot hybridization for the presence of DNA that hybridized to a pUC19 probe. This screening methodology assured that all clones, even clones lacking an insert but yet containing the pWE1 5 plasmid, would be detected.
Liquid cultures of all 189 transformants were used to generate cosmid minipreps for analysis of restriction sites within the insert DNA.
Six of the original 189 cosmid clones contained an insert. These clones were designated as follows: 28 (-9-kb insert), 30 (- 9-kb insert), 60 (- 4-kb insert), 113 (-- 9-kb insert), 157 (-- 9-kb insert) and 161 (-- 9-kb insert). Restriction enzyme analysis indicated that three of the clones (113, 157 and 161) contained the same insert.
For sequence analysis the insert of cosmid clone no. 161 was subcloned as follows. To obtain the end fragments of the insert of clone no. 161, the clone was digested with Not] and BamHl and ligated with Notl/BamHl-digested pBluescript KS (Stratagene, La Jolla, California).
Two fragments of the insert of clone no. 161 were obtained: a 0.2-kb and a 0.7-kb insert fragment. To subclone the internal fragment of the insert of clone no. 161, the same digest was ligated with BamHl-digested pUC1 9. Three fragments of the insert of clone no. 161 were obtained: a 0.6-kb, a 1.8-kb and a 4.8-kb insert fragment.
The insert corresponds to an internal section of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 as set forth in GENBANK accession no. X82564, which is provided as SEQ ID NO. 18. The sequence data obtained for the insert of clone no.
161 is set forth in SEQ ID NOS. 19-25. Specifically, the individual subclones corresponded to the following positions in GENBANK accession no. X82564 (SEQ ID NO:18) and in SEQ ID NOs. 19-25:

=05-56(S) Subclone Start End Site SEQ ID No.
in X82564 161k1 7579 7755 Notl, BamHl 19 161m5 7756 8494 BamHl 20 161m7 8495 10231 BamHl 21 (shows only sequence corresponding to nt. 8495-8950), 22 (shows only sequence corresponding to nt. 9851- 10231) 161m12 10232 15000 BamHl 23 (shows only sequence corresponding to nt. 10232-10600), 24 (shows only sequence corresponding to nt. 14267-15000) 161k2 15001 15676 Notl, BamHt 25 The sequence set forth in SEQ ID NOs. 19-25 diverges in some positions from the sequence presented in positions 7551-15670 of GENBANK accession no. X82564. Such divergence may be attributable to random mutations between repeat units of rDNA.
For use herein, the rDNA insert from the clone was prepared by digesting the cosmid with Notl and Bglll and was purified as described above. Growth and maintenance of bacterial stocks and purification of plasmids were performed using standard well known methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press), and plasmids were purified from bacterial cultures using Midi - and Maxi-preps Kits (Qiagen, Mississauga, Ontario).
pDsRed1N1 This vector is available from Clontech*(see SEQ ID No. 29) and encodes the red fluorescent protein (DsRed; Genbank accession no.
AF272711; SEQ ID Nos. 39 and 40). DsRed, which has a vivid red fluorescence, was isolated from the IndoPacific sea anemone relative Discosoma species. The plasmid pDsRed1N1 (Clontech; SEQ ID No. 29) constitutively expresses a human codon-optimized variant of the *Trade-mark fluorescent protein under control of the CMV promoter. Unmodified, this vector expresses high levels of DsRedl and includes sites for creating N-terminal fusions by cloning proteins of interest into the multiple cloning site (MCS). It is Kan and Neo resistant for selection in bacterial or eukaryotic cells.
Plasmid pMG
Plasmid pMG (InvivoGen, San Diego, California; see SEQ. ID. NO.
27 for the nucleotide sequence of pMG) contains the hygromycin phosphotransferase gene under the control of the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A. Vector pMG
also contains two transcriptional units allowing for the coexpression of two heterologous genes from a single vector sequence.
The first transcriptional unit of pMG contains a multiple cloning site for insertion of a gene of interest, the hygromycin phosphotransferase gene (hph) and the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A (see, e.g., Chapman et al. (1991) Nuc.
Acids Res. 19:3979-3986) located upstream of hph and the multiple cloning site, which drives the expression of hph and any gene of interest inserted into the multiple cloning site as a polycistronic mRNA. The first transcriptional unit also contains a modified EMCV internal ribosomal entry site (IRES) upstream of the hph gene but downstream of the hCMV
promoter and MCS for ribosomal entry in translation of the hph gene (see SEQ ID NO. 27, nucleotides 2736-3308). The IRES is modified by insertion of the constitutive E. coil promoter (EM7) within an intron (IM7) into the end of the IRES. In mammalian cells, the E. coil promoter is treated as an intron and is spliced out of the transcript, A
polyadenylation signal from the bovine growth hormone (bGh) gene (see, e.g., Goodwin and Rottman (1992) J. Biol. Chem. 267:16330-16334) and a pause site derived from the 3' flanking region of the human a2 globin gene (see, e.g., Enriquez-Harris et al. (1991) EMBO J.10:1833-1842) are located at the end of the first transcription unit. Efficient polyadenylation is facilitated by inserting the flanking sequence of the bGh gene 3' to the standard AAUAAA hexanucleotide sequence.
The second transcriptional unit of pMG contains another multiple cloning site for insertion of a gene of interest and an EF-1 a/HTLV hybrid promoter located upstream of this multiple cloning site, which drives the expression of any gene of interest inserted into the multiple cloning site.
The hybrid promoter is a modified human elongation factor-1 alpha (EF-1 alpha) gene promoter (see, e.g., Kim et al. (1990) Gene 91:217-223) that includes the R segment and part of the U5 sequence (R-U5') of the human T-cell leukemia virus (HTLV) type I long terminal repeat (see, e.g., Takebe et al. (1988) Mol. Cell. Biol 8:466-472). The Simian Virus 40 (SV40) late polyadenylation signal (see Carswell and Alwine (1989) Mol.
Cell. Biol. 9:4248-4258) is located downstream of the multiple cloning site. Vector pMG contains a synthetic polyadenylation site for the first and second transcriptional units at the end of the transcriptional unit based on the rabbit /3-globin gene and containing the AATAAA
hexanucleotide sequence and a GT/T-rich sequence with 22-23 nucleotides between them (see, e.g., Levitt et al. (1989)Genes Dev.
3:1019-1025). A pause site derived from the C2 complement gene (see, Moreira et al. (1995) EMBO J. 14:3809-3819) is also located at the 3' end of the second transcriptional unit.
Vector pMG also contains an on sequence (ori pMB1) located between the SV40 polyadenylation signal and the synthetic polyadenylation site.

A. Construction of targeting vector and transfection into LMtk- cells for the generation of platform chromosomes A targeting vector derived from the vector pWE15 (GeneBank Accession # X65279) was modified by replacing the Sall (Kienow filled)/Smal neomycin resistance containing fragment with the PvulI/BamHl (Klenow filled) puromycin resistance containing fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc. Palo Alto, CA;
SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb Notl fragment from the plasmid pFK1 61 (SEQ ID NO: 118) containing a portion of the mouse rDNA region was cloned into the Notl site of pWEPuro resulting in plasmid pWEPuro9K (Figure 2). The vector pWEPuro9K was digested with Spel to linearize and transfected into LMtk- mouse cells.
Puromycin resistant colonies were isolated and subsequently tested for artificial chromosome formation via fluorescent in situ hybridization (FISH) (using mouse major and minor DNA repeat sequences, the puromycin gene and telomeres sequences as probes), and fluorescent activated cell sorting (FACS). From this sort, a subclone was isolated containing an artificial chromosome, designated 5131 1.12, which carries 4-8 copies of the puromycin resistance gene contained on the pWEPuro9K vector.
FISH analysis of the 51311. 12 subclone demonstrated the presence of telomeres and mouse minor on the ACes. DOT PCR has been done on the 51311.12 ACes revealing the absence of uncharacterized euchromatic regions on the ACes. A recombination site, such as an att or loxP
engineering site or a plurality thereof, was introduced onto this ACes thereby providing a platform for site-specific introduction of heterologous nucleic acid.
B. Targeting a single sequence specific recombination site onto platform chromosomes After the generation of the 51311. 12 platform, a single sequence-specific recombination site is placed onto the platform chromosome via homologous recombination. For this, DNA sequences containing the site-=

specific recombination sequence can be flanked with DNA sequences of homology to the platform chromosome. For example, using the platform chromosome made from the pWEPuro9K vector, mouse rDNA sequences or mouse major satellite DNA can be used as homologous sequences to target onto the platform chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the platform chromosome, the DNA is transfected into cells harboring the platform chromosome (Figure 3). Examples of site-specific cassettes that are targeted to the platform chromosome using either mouse rDNA or mouse major repeat DNA include the SV40-attP-hygro cassette and a red fluorescent protein (RFP) gene flanked by loxP sites (Cre/lox, see, e.g., U.S. Patent No. 4,959,317 and description herein).
After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified by FACS (e.g. screen and single cell subclone via expression of resistance or fluorescent marker) and PCR analysis.
For example, a vector can be constructed containing regions of the mouse rDNA locus flanking a gene cassette containing the SV40 early reporter-bacteriophage lambda attP site-hygromycin selectable marker (see Figure 4 and described below). The use of the bacteriophage lambda attP site for lambda integrase-mediated site-specific recombination is described below. Homologous recombination event of the SV40-attP-hygro cassette onto the platform chromosome was identified using PCR
primers that detect the homologous recombination and further confirmed by FISH analysis. After identifying subcloned colonies containing the platform chromosome with a single site-specific recombination site, cells carrying the platform chromosome with a single site-specific =

recombination site can now be engineered with site-specific recombinases (e.g. lambda INT, Cre) for integrating a target gene expression vector.

C. Targeting a red fluorescent protein (RFP) gene flanked by loxP sites onto 5B11.12 platform As another example, while loxP recombination sites could have been introduced onto the ACes during de novo biosynthesis, it was thought that this might result in multiple segments of the ACes containing a high number of loxP sites, potentially leading to instability upon Cre-mediated recombination. A gene targeting approach was therefore devised to introduce a more limited number of loxP recombination sites into a locus of the 5B11-12 ACes containing introduced and possibly co-amplified endogenous rDNA sequences. Although there are more than 200 copies of rDNA genes in the haploid mouse genome distributed amongst 5-1 1 chromosomes (depending on strain), rDNA sequences were chosen as the target on the ACes since they represent a less frequent target than that of the satellite repeat sequences. Moreover, having observed much stronger pWEPuro9K hybridization to the 5B11-12 ACes than to other LMTK" chromosomes and in light of the observation that the transcribed spacer sequences within the rDNA may be less conserved than the rRNA coding regions, it was contemplated that a targeting vector based on the rDNA gene segment in pWEPuro9K would have a higher probability of targeting to the ACes rather than to other LMTK-chromosomes. Accordingly, a targeting vector, pBSFKLoxDsRedLox, was designed and constructed based on the rDNA sequences contained in pWEPuro9K.
The plasmid pBSFKLoxDsRedLox was generated in 4 steps. First, the Notl rDNA insert of pWEPuro9K (Figure 2) was inserted into pBS SK-(Stratagene) giving rise to pBSFK. Second, a loxP polylinker cassette was = .

generated by PCR amplification of pNEB193 (SEQ ID NO:32; New England Biolabs) using primers complementary to the M13 forward and reverse priming sites at their 3'end and a 34 bp 5' extension comprising a LoxP site. This cassette was reinserted into pNEB1 93 generating pl 93LoxMCSLox. Third, the DsRed gene from pDsRedl-N1 (SEQ ID
NO:29; Clontech) was then cloned into the polylinker between the loxP
sites generating p193LoxDsRedLox. Fourth, a fragment consisting of the DsRed gene flanked by loxP sites was cloned into a unique Ndel within the rDNA insert of pBSFK generating pBSFKLoxDsRedLox.

A gel purified 11 Kb Pm/I /EcoRV fragment of pBSFKLoxDsRedLox was used for transfection. To detect targeted integration, PCR primers were designed from rDNA sequences within the 5' Notl-Pmll fragment of pWEPuro9K that is not present on the targeting fragment (5'primer) and sequence within the LoxDsRedLox cassette (3' primer). If the targeting DNA integrated correctly within the rDNA sequences, PCR amplification using these primers would give rise to a 2.3 Kb band. PCR reactions containing 1-4 ,u1 of genomic DNA were carried out according to the MasterTaq protocol (Eppendorf), using murine rDNA 5' primer (5'-CGGACAATGCGGTTGTGCGT-3'; SEQ ID NO:72) and DsRed 3'primer (5'GGCCCCGTAATGCAGAAGAA-3'; SEQ ID NO:73) and PCR products were analyzed by agarose gel electrophoresis.
1.5X 106 5B11-12 LMTK" cells were transfected with 2 jig of the pBSFKLoxDsRedLox targeting DNA described above using Lipofectamine Plus (Invitrogen). For flow sorting, harvested cells were suspended in medium and applied to the Becton Dickinson Vantage SE cell sorter, equipped with 488 nm lasers for excitation and 585/42 bandpass filter for optimum detection of RFP fluorescence. Cells were sorted using dPBS as sheath buffer. Negative control parental 5B11-12 cells and a positive control LMTK- cell line stably transfected with DsRed were used to establish the selection gates. The RFP positive gated populations were recovered, diluted in medium supplemented with 1X penicillin-streptomycin (Invitrogen), then plated and cultured as previously described. After 4 rounds of enrichment, the percentage of RFP positive cells reached levels of 50% or higher. DNA from populations was analyzed by PCR for evidence of targeted integration. Ultimately, single cell .subclones were established from positive pools and were analyzed by PCR and PCR-positive clones confirmed by FISH as described below.
DNA was purified from pools or single cell clones using previously described methods set forth in Lahm et al., Transgenic Res., 1998;
7:131-134, or in some cases using a Wizard Genomic DNA purification kit (Promega). For FISH analysis, a biotinylated DsRed gene probe was generated by PCR using DsRed specific primers and biotin-labeled dUTP
(5' RFP primer: 5'-GGTTTAAAGTGCGCTCCTCCAAGAACGTCATC-3', SEQ ID NO:74; and 3' RFP primer:
5'AGATCTAGAGCCGCCGCTACAGGAACAGGTGGTGGCGGCC-3'; SEQ
ID NO:75). To maximize the signal intensity of the DsRed probe, Tyramide amplification was carried out according to the manufacturers protocols (NEN).
The process of testing the feasibility of a more general targeting strategy that would not rely on enrichment via drug selection of stably transfected clones can be summarized as follows. A red fluorescent protein gene (RFP; encoded by the DsRed gene) was inserted between the IoxP sites of the targeting vector to form pBSFKLoxDsRedLox. After transfection with PBSFKLoxDsRedLox, sequential rounds of high speed flow sorting and expansion of sorted cells in culture could then be used to enrich for stable transformants expressing RFP. In the event of targeted integration, PCR screening with primers that amplify from a spacer region within the segment of the 45s pre-rRNA gene in pWEPuro9K to a specific = S' anchor sequence within the DsRed gene in the targeting cassette would give rise to a diagnostic 2.3 Kb band. However, as rDNA clusters are found on several chromosomes, confirmation of targeting to an ACes would require fluorescence in situ hybridization (FISH) analysis. Finally, the flanking of the DsRed gene by IoxP sites would allow for its removal and subsequent replacement with other genes of interest.
After transfection of the targeting sequence into 5811-12 cells, enrichment for targeted clones was carried out using a combination of flow cytometry to detect red-fluorescing cells and PCR screening.
Ultimately 17 single cell subclones were identified as potential targeted clones by PCR and of these 16 were found by FISH to contain the DsRed integration event into the ACes. These subclones are referred to herein as DI 1 -C4, D1 1-C12, D1 1 -H3, C9-C9, C9-B9, C9-F4, C9-H8, C9-F2, C9-G8, C9-B6, C9-G3, C9-E12, C9-A11, C11-E3, C11-A9 and C11-H4. PCR
analysis of genomic DNA isolated from the D11-C4 subclone gave rise to a 2.3 Kb band, indicative of a targeted integration into an rDNA locus.
Further analysis of the subclone by FISH analysis with a DsRed gene probe demonstrated integration of the LoxDsRedLox targeting cassette on the ACes co-localizing with one of the regions of rDNA staining seen on the 51311-12 ACes, consistent with a targeted integration into an rDNA
locus of the ACes, while integrations on other chromosomes were not observed. Since transfected cells were maintained as heterogeneous populations through several cycles of sorting and replating it was not possible to estimate the frequency of targeted events. In most mammalian cell lines the frequency of gene targeting via homologous recombination is roughly 10'5-10,7 treated cells. Despite the low frequency of these events in mammalian cells, it is clear that an RFP
expression based screening paradigm, coupled with PCR analysis, can effectively detect and enrich for such infrequent events in a large population. In instances where drug selection is not possible or not desirable, such a system may provide a useful alternative. It was also verified that the modified ACes in subclone D11-C4 could be purified by flow cytometry. The results indicate that the flow karyogram of the D11-C4 subclone was unaltered from that of the 5611-12 cell line. Thus, the D11-C4 ACes can be purified in high yield from native chromosomes of the host cell line.

D. Reduction of LoxP on ACes to a single site.
The strong hybridization signal detected by FISH on the ACes using the DsRed gene probe suggests that several copies of the targeting cassette may be present on the ACes in the D11-C4 line. This also suggests that multiple rDNA genes have been correctly targeted.
Accordingly, in certain embodiments where necessary, the number of loxP sites on the ACes can be reduced to a single site by in situ treatment with Cre recombinase, provided that the sites are co-linear.
Such a process is described for multiple loxP-flanked integrations on a native mouse chromosome (Garrick et al., Nature Genet., 1998, Jan;18(1):56-59). Reduction to a single loxP site on the D11-C4ACes would result in the loss of the DsRed gene, forming the basis of a useful screen for this event.
For this purpose, a Cre expression plasmid pCX-Cre/GFP III has been generated by first deleting the EcoRl fragment of pCX-eGFP (SEQ ID
N0:71) containing the eGFP coding sequence and replacing it with that of a PCR amplified Cre recombinase coding sequence (SEQ ID N0:58), generating pCX-Cre. Next, the Asel/Sspl fragment of pD2eGFP-N1 (containing the CMV promoter driving the D2EGFP gene with SV40 polyA
signal; Clontech; SEQ ID NO:87) was inserted into the filled Hindlll site of pCX-Cre, generating pCX-Cre\GFP lll. Control plasmid pCX-CreRev\GFP

III was generated in similar fashion except that the Cre recombinase coding sequence was inserted in the antisense orientation. LMTK- cell line D1 1-C4 (containing first generation platform ACes with multiple loxP-DsRED sites) and 5B11-12 cell line (containing ACes with no IoxP-DsRED
sites) are maintained in culture as described above. D11 C4 cells are transfected with 2,ug of plasmid pCX-Cre\GFP III or 2 Ng pCX-CreRev\GFP III using Lipofectamine (lnvitrogen) as previously described.
Forty-eight to seventy-two hours after transfection, transfected D11-C4 cells are harvested and GFP positive cells are sorted by cell cytometry using a FACSta Vantage cell sorter (Beckton-Dickinson) as follows: All D11-C4 cells transfected with pCX-Cre\GFP III or control plasmid pCX-CreRev\GFP III that exhibit GFP fluorescent higher than the gate level established by untransfected cells are collected and placed in culture a further 7-14 days. After 7-14 days the initial D11-C4 cells are harvested and analyzed by cell cytometry as follows: Untransfected D11-C4 cells are used to establish the gate that defines the RFP positive population, while 5B11-12 cells are used to set the RFP negative gate.
The GFP positive population of D1 1-C4 transfected with pCX-Cre\GFP III
should show decreased red fluorescence compared to pCX-CreRev\GFP III
transfected or untransfected control D11-C4 cells. The cells exhibiting greatly decreased or no RFP expression are collected and single cell clones subsequently established. These clones will be expanded and analyzed by fluorescence in-situ hybridization and Southern blotting to confirm the removal of IoxP-DsRed gene copies.

Construction of targeting vector and transfection into LMtk- cells for the generation of platform chromosomes containing multiple site-specific recombination sites An example of a selectable marker system for the creation of a chromosome-based platform is shown in Figure 4. This system includes a vector containing the SV40 early promoter immediately followed by (1) a 282 base pair (bp) sequence containing the bacteriophage lambda attP
site and (2) the puromycin resistance marker. Initially a Pvull/Stul fragment containing the SV40 early promoter from plasmid pPUR
(Clontech Laboratories, Inc., Palo Alto, CA; Seq ID No. 30) was subcloned into the EcoRIICRI site of pNEB1 93 (a PUC1 9 derivative obtained from New England Biolabs, Beverly, MA; SEQ ID No. 32) generating the plasmid pSV40193. The only differences between pUC19 and pNEB1 93 are in the polylinker region. A unique Ascl site (GGCGCGCC) is located between the BamHl site and the Smal site, a unique Pacl site (TTAATTAA) is located between the BamHl site and the Xbal site and a unique Prnel site (GTTTAAAC) is located between the Pstl site and the Sa/I site.
The attP site was PCR amplified from lambda genome (GenBank Accession # NC 001416) using the following primers:
attPUP: CCTTGCGCTAATGCTCTGTTACAGG SEQ ID No. 1 attPDWN: CAGAGGCAGGGAGTGGGACAAAATTG SEQ ID No. 2 After amplification and purification of the resulting fragment, the attP site was cloned into the Smal site of pSV40193 and the orientation of the attP site was determined by DNA sequence analysis (plasmid pSV40193attP). The gene encoding puromycin resistance (Puro) was isolated by digesting the plasmid pPUR (Clontech Laboratories, Inc. Palo Alto, CA) with Agel/BamHl followed by filling in the overhangs with Klenow and subsequently cloned into the Ascl site downstream of the attP site of pSV40193attP generating the plasmid pSV40193attPsensePUR (Figure 4; SEQ ID NO:1 13)).

= ~' The plasmid pSV40193attPsensePUR was digested with Scal and co-transfected with the plasmid pFK161 (SEQ ID NO: 118) into mouse LMtk- cells and platform artificial chromosomes were identified and isolated as described above. The process for generating this exemplary platform ACes containing multiple site-specific recombination sites is summarized in Figure 5. One platform ACes resulting from this experiment is designated B19-18. This platform ACes chromosome may subsequently be engineered to contain target gene expression nucleic acids using the lambda integrase mediated site-specific recombination system as described herein in Example 7 and 8.

Lambda integrase mediated site-specific recombination of a RFP
expressing vector onto artificial chromosomes In this example, a vector expressing the red fluorescent protein (RFP) was produced and recombined into the attP site residing on an artificial chromosome within LMTK- cells. This recombination is depicted in Figure 7.
A. Construction of expression vectors containing wildtype and mutant lambda integrase Mutations at the glutamic acid at position 174 in the lambda integrase protein relaxes the requirement for the accessory protein IHF
during recombination and DNA supercoiling in vitro (see, Miller et al.
(1980) Ce/I 20:721-729; Lange-Gustafson et al. (1984) J. Biol. Chem.
259:12724-12732). Mutations at this site promote attP, attB
intramolecular recombination in mammalian cells (Lorbach et al. (2000) J.
Mol. Biol 296:1175-1 181).
To construct nucleic acid encoding the mutant, lambda integrase was PCR amplified from bacteriophage lambda DNA (c1857 ind Sam 7;
New England Biolabs) using the following primers:
Lamintl (SEQ ID No. 3) =

TTCGAATTCATGGGAAGAAGGCGAAGTCATGAGCG) Lamint2 (SEQ ID No. 4) (TTCGAATTCTTATTTGATTTCAATTTTGTCCCAC).
The resulting PCR product was digested with EcoR I and cloned into the EcoR I site of pUC19. Lambda integrase was mutated at amino acid position 174 using QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligos (generating a glutamic acid to arginine change at position 174):
Lambda I NTE1 74R
(SEQ ID No. 6) (CGCGCAGCAAAATCTAGAGTAAGGAGATCAAGACTTACGGCTGACG), LamintR174rev (SEQ ID No. 7) (CGTCAGCCGTAAGTCTTGATCTCCTTACTCTAGATTTTGCTGCGCG).
The resulting site directed mutant was confirmed by sequence analysis.
The wildtype and mutant lambda genes were cloned into the EcoR I site of pCX creating pCX-Lamlnt (SEQ ID NO: 127) and pCXLamIntR (Figure 8; SEQ ID NO: 112).
The plasmid pCX (SEQ ID No. 70) was derived from plasmid pCXeGFP (SEQ ID No. 71). Excision of the EcoRl fragment containing the eGFP marker generated pCX. To generate plasmid pCXLamINTR (SEQ ID
NO: 112) an EcoRl fragment containing the lambda integrase El 74R (SEQ
ID No. 37) mutation was cloned into the EcoRl site of pCX, and to generate plasmid pCX-LamINT, an EcoRl fragment containing the wild-type lambda integrase was cloned into the EcoRl site of pCX.
B. Construction of integration vector containing attB and DsRed The plasmid pDsRedN1 (Clontech Laboratories, Palo Alto, CA; SEQ
ID No. 29) was digested with Hpa I and ligated to the following annealed oligos:
attB1 (SEQ ID No. 8) =

(TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA) attB2 (SEQ ID No. 9) (TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA) The resulting vector (pDsRedNl-attB) was confirmed by PCR and sequence analysis.
C. Transfection into LMtk- cells LM(tk-) cells containing the Prototype AACes (L1-18; Chromos Molecular Systems Inc., Burnaby, BC Canada) were co-transfected with pDsRedN1 or pDsRedNl-attB and either pCXLamint (SEQ ID NO: 127) or pCXLamlntR (SEQ ID NO: 112) using Lipofectamine Plus Reagent (LifeTechnologies, Gaithersburg, MD). The transfected cells were grown in DMEM (LifeTechnologies, Gaithersburg, MD) with 10% FBS (CanSera) and G418 (CalBiochem) at a concentration of 1 mg/ml.
D. Enrichment by cell sorting The transfected cells were sorted using a FACs Vantage SE cell sorter (Becton Dickenson) to enrich for cells expressing DsRed. The cells were excited with a 488 nm Argon laser at 200 watts and cells fluorescing in the 585/42 detection channel were collected. The sorted cells were returned to growth medium for recovery and expansion. After three successive enrichments for cells expressing DsRed, single cell sorting into 96 well plates was performed using the same parameters.
Duplicate plates of the single cell clones were made for PCR analysis.
E. PCR analysis of single cell clones Pools of cells from each row and column of the 96 well plate were used for DNA isolation. DNA was prepared using a Wizard Genomic DNA
purification kit (Promega Inc, Madison, WI). Nested PCR analysis on the DNA pools was performed to confirm the site-specific recombination event using the following primer sets:

attPdwn2 (SEQ ID No. 10) (TCTTCTCGGGCATAAGTCGGACACC) CMVen (SEQ ID No. 11) (CTCACGGGGATTTCCAAGTCTCCAC) followed by:
attPdwn (SEQ ID No. 12) (CAGAGGCAGGGAGTGGGACAAAATTG) CMVen2 (SEQ ID No. 13) (CAACTCCGCCCCATTGACGCAAATG).
The resulting PCR reactions were analyzed by gel electrophoresis and the potential individual clones containing the site-specific recombination event were identified by combining the PCR results of all of the pooled rows and columns for each 96 well plate. The individual clones were then further analyzed by PCR using the following primers that flank the recombination junction. L1 for and F1 rev flank the attR junction whereas REDfor and L2rev flank the attL junction (see Figure 7):
L1 for (SEQ ID No. 14) AGTATCGCCGAACGATTAGCTCTTCA
F1 rev (SEQ ID No. 15) GCCGATTTCGGCCTATTGGTTAAA
REDfor (SEQ ID No. 16) CCGCCGACATCCCCGACTACAAGAA
L2rev (SEQ ID No. 17) TTCCTTCGAAGGGGATCCGCCTACC.
F. Sequence analysis of recombination junctions PCR products spanning the recombination junction were Topo-cloned into pcDNA3.1 D/V5His (Invitrogen Inc., San Diego, CA) and then sequenced by cycle-sequencing. The clones were confirmed to have the correct attR and attL junctions by cycle sequencing.
G. Fluorescent In Situ Hybridization (FISH) The cell lines containing the correct recombination junction sequence were further analyzed by fluorescent in situ hybridization (FISH) = =

by probing with the DsRed coding region labeled with biotin and visualizing with the Tyramide Signal Amplification system (TSA; NEN Life Science Products). The results indicate that the RFP sequence is present on the ACes.
H. Southern analysis Genomic DNA was harvested from the cell lines containing an ACes with the correct recombinant event and digested with EcoR I. The digested DNAs were separated on a 0.7% agarose gel, transferred and fixed to a nylon membrane and probed with RFP coding sequences. The result showed that there is an integrated copy of RFP coding sequence in each clone.

Delivery of a second gene encoding GFP onto the RFP platform ACes A. Construction of integration vector containing attB and GFP
(pD2eGFPlresPuroattB).
The plasmid pIRESpuro2 (Clontech, Palo Alto, CA; SEQ ID NO: 88) was digested with EcoRI and Not[ then ligated to the D2eGFP EcoRl-Notl fragment from pD2eGFP-N1 (Clontech, Palo Alto, CA) to create pD2eGFPlresPuro2. Subsequently, oligos encoding the attB site were annealed and ligated into the Nrul site of pD2eGFPlresPuro2 to create pD2eGFPlresPuroattB. The orientation of attB in the Nrul site was determined by PCR.
B. Transfection of LMtk- cells The LMtk- cells containing the RFP platform ACes produced in Example 4, which has multiple attP sites, were co-transfected with pCXLamlntR and pD2eGFPlresPuroattB using LipofectAMINE PLUS
reagent. Five jig of each vector was placed into a tube containing 750,41 of DMEM (Dulbecco's modified Eagles Medium). Twenty /A of the Plus reagent was added to the DNA and incubated at room temperature for 15 '005-56(s) minutes. A mixture of 30 NI of lipofectamine and 750 pI DMEM was added to the DNA mixture and incubated an additional 15 minutes at room temperature. The DNA'mixture was then added dropwise to approximately 3 million cells attached to a 10cm dish in 5 mis of DMEM.
The cells were incubated 4 hours (37 C, 5% C02) with the DNA-lipid mixture, after which DMEM with 20% fetal bovine serum was added to the dishes to bring the culture medium to 10% fetal bovine serum. The dishes were incubated at 37 C with 5% CO2.
Plasmid pD2eGFPlresPuroattB has a puromycin gene transcriptionally linked to the GFP gene via an IRES element. Two days after the transfection the cells were placed in medium containing puromycin at 4,ug/ml to select for cells containing the pD2eGFPlresPuroattB plasmid integrated into the genome. Twenty-three clones were isolated after 17 days of selection with puromycin. These clones were expanded and then analyzed for the presence of the GFP
gene on the ACes by 2-color (RFP/biotin & GFP/digoxigenin) TSA-FISH
(NEN) according to the manufacturers protocol. Sixteen of the 23 clones produced a positive FISH signal on the ACes with a GFP probe.

Delivery Of ACes Into human Mesenchymal Stem Cells (hMSC) A. Transfection Transfection conditions for the most efficient delivery of the ACes into hMSCs (Cambrex BioWhittaker Product Code PT-2501, lot# F0658, East Rutherford, New Jersey) were assayed, using -LipofectAMINE PLUSH
and Superfect* One million prototype B ACes, which is a murine derived 60Mb ACes having primarily murine pericentric heterochromatin, and carrying a "payload" containing a hygromycin B selectable marker gene and a lacZ reporter gene (see , Telenius et at., 1999, Chrom. Res., 7:3-7;
and Kereso at at., 1996, Chrom. Res., 4:226-239; each of which is *Trade-mark incorporated herein by reference in its entirety), were combined with 1-12 Nl of the transfection agent. In the case of LipofectAMINE PLUS, the PLUS reagent was combined with the ACes for 15 minutes followed by LipofectAMINE for a further 15 minutes. Superfect was complexed for 10 minutes at a ratio of 2Nl Superfect per 1 million ACes. The ACes/transfection agent complex was then applied to 0.5 million recipient cells and the transfection was allowed to proceed according to the manufacturer's protocol. Percent transfected cells was determined on a FACS Vantage flow cytometer with argon laser tuned to 488 nm at 200mW and FITC fluorescence collected through a standard FITC 530/30 nm band pass filter. After 24 hours, IdUrd labeled ACes were delivered to human MSCs in the range of 30-50%, varying with transfection agent and dose. ACes delivery curves were generated from data collected in experiments that varyied the dose of the transfection reagents. Dose response curves of Superfect and LipofectAMINE PLUS, showing delivery of ACes into recipient hMSCs cells, were prepared, measured by transfer of IdUrd labeled ACes and detected by flow cytometry. Superfect shows maximum delivery in the range of 30-50% at doses greater than 2 Ni per million ACes. LipofectAMINE PLUS has a 42-48% delivery peak around 5-8 pi per million ACes. These dose curves were then correlated with toxicity data to determine the transfection conditions that will allow for highest potential transfection efficiency. Toxicity was determined by a modified plating efficiency assay (de Jong et at., 2001, Chrom. Research, 9:475-485). The population's normalized plating efficiency (at maximum % delivery doses) was in the range of 0.2 - 0.4 for Superfect and 0.5 -0.6 with LipofectAMINE PLUS.

Due to the transfected population consisting of mixed cell types, flow cytometry allowed for the assessment of ACes delivery into each sub-population and the purification of the target population. Flow profiles =

showing forward scatter (cell size) and side scatter (internal cell granularity) revealed three distinct hMSC populations that were gated into three regions: R3 (small cell region), R4 (medium cell region), R5 (large cell region). Transfection conditions were further optimized by re-analyzing delivery curves and assessing the differences in delivery to each sub-population. Dose response curves of Superfect and LipofectAMINE
were prepared showing % delivery to each sub-population represented by the gating on basis of cell size and granularity properties of the mixed population. Three distinct hMSC populations were gated and % delivery dose curves generated. Using Superfect and LipofectAMINE PLUS the overall % delivery increased with cell size (80-90% delivery in large cells).
LipofectAMINE PLUS at high doses (8-12 NI per 1 million ACes) shows an increase in the overall proportion of chromosome transfer to the small population (10-20%). This suggests an advantage to using this transfection agent if the small-undifferentiated cell population is the desired target host cell.
B. Expression from Genes on Aces IN hMSCs Following the delivery screening process conducted in section (A) above, the most promising results were subjected to further analyses to monitor expression and verify the presence of structurally intact ACes.
The transfection conditions employed for these experiments were exactly the same as those that had been used during the screening process.
Short-term expression was monitored by transfecting hMSCs with ACes containing a RFP gene (red fluorescent protein) set forth in Example 2C as "D1 1C4". The unselected population was harvested at 72-96 hours post transfection and % positive fluorescent cells measured by flow cytometry. RFP expression was in the range of 1-20%.
Long term-gene expression was assayed by selecting for hygromycin B resistant cells over a period of 7-10 days. Cytogenetic = .

analysis was done to detect presence of intact ACes by Fluorescent In Situ hybridization (FISH), where metaphase chromosomes were hybridized to a mouse major satellite-DNA probe (targeting murine pericentric heterochromatin) and a lambda probe (hybridizing to the /acZ gene). The human mesenchymal transfected culture could not undergo standard sub-cloning as diffuse colonies form with limited doublings available for expansion. Cytogenetic analysis was performed on the entire population, sampling over a period of 3-10 days post-transfection. The hygromycin resistant population was then blocked in mitosis with colchicine and analyzed for presence of intact ACes by FISH. Preliminary FISH results show approximately 2-8% of the hMSC-transfected population had an intact ACes. This compared to rat skeletal muscle myoblast clones, which were in the range of 60-95%. To increase the % of intact ACes in the hMSC-transfected population an enrichment step can be utilized as described in Example 2C.
C. Differentiation of The hMSCs In initial experiments where transfected hMSCs cells have been induced to differentiate into adipose or osteocytes, the results indicate that the transfected cells appear to be differentiating at a rate comparable to the untransfected controls and the cultures are lineage specific as tested by microscopic examination, FISH, Oil Red 0 staining (adipocyte assay), and calcium secretion (osteocyte assay).
Accordingly, these results indicate that the artificial chromosomes (ACes) provided herein can be successfully transferred into hMSC target cells. Targeting MSCs (such as hMSCs) permits gene transfer into cells in an undifferentiated state where the cells are easier to expand and purify.
The genetically modified cells can then be differentiated in vitro or injected into a site in vivo where the microenvironment will induce transformation into specific cell lineages.

Delivery of a Promoterless Marker Gene to a Platform ACes Platform ACes containing pSV40attPsensePURO (Figure 4) were constructed as set forth in Examples 3 and 4.
A. Construction of Targeting Vectors.

The base vector pl8attBZeo (3166bp; SEQ ID NO: 114) was constructed by ligating the 1067bp Hindlll-Sspl fragment containing attBZeo, obtained from pLlTattBZeo (SEQ ID NO:91), into pUC18 (SEQ ID
NO: 122) digested with Hindlll and Sspl.

1. p18attBZEO-eGFP (6119bp; SEQ ID NO: 126) was constructed by inserting the 2977bp Spel-Hindlll fragment from pCXeGFP (SEQ ID
NO:71; Okabe, et al. (1997) FEBS Lett 407:313-319) containing the eGFP
gene into p18attBZeo (SEQ ID NO: 114) digested with Hindlll and Xbal.
2. pl 8attBZEO-5'6XHS4eGFP (Figure 10; 7631 bp; SEQ ID NO:
116) was constructed by ligating the 4465bp Hindlll fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123), which contains the eGFP gene under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5' of the chicken beta actin promoter and the polyadenylation signal, into the Hindlll site of p18attBZeo (SEQ ID NO:
114).

3. p18attBZEO-3'6XHS4eGFP (Figure 11; 7600bp; SEQ ID NO:
115) was created by removing the 5'6XHS4 element from p 1 8attBZeo-(6XHS4)2eGFP (SEQ ID NO: 110). pl8attBZeo-(6XHS4)2eGFP was digested with EcoRV and Spel, treated with Klenow and religated to form pl8attBZeo3'6XHS4eGFP (SEQ ID NO: 115).

4. pl8attBZEO-(6XHS4)2eGFP (Figure 12; 9080bp; SEQ ID NO:
110) was created in two steps. First, the EcoRl-Spel fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123), which contains 6 copies of the HS4 core element, was ligated into p18attBZeo (SEQ ID NO: 114) Rectified Bice (Rule 91) = =

digested with EcoRl and Xbal to create pl8attBZeo6XHS4 (4615bp; SEQ
ID NO: 117). Next, pl8attBZeo6XHS4 was digested with HindIll and ligated to the 4465bp HindIll fragment from pCXeGFPattB(6XHS4)2 which contains the eGFP gene under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5' of the chicken beta actin promoter and the polyadenylation signal.
Table 2 Targeting plasmid No. zeocin No. clones with No. clones with correct resistant expected PCR sequence at clones product size recombination junction pl 8attBZEO-eGFP 12 12 NT*

pl8attBZEO-5'6XHS4eGFP 11 11 NT
pl8attBZEO-3'6XHS4eGFP 11 11 NT
p18attBZEO-(6XHS4)2eGFP 9 9 4/4 *NT = not tested B. Transfection and Selection with Drug.
The mouse cell line containing the 2"d generation platform ACE, B19-38 (constructed as set forth in Example 3), was plated onto four 10cm dishes at approximately 5 million cells per dish. The cells were incubated overnight in DMEM with 10% fetal calf serum at 37 C and 5%
CO2. The following day the cells were transfected with 5,ug of each of the 4 vectors listed in Example 7.A. above and 5pg of pCXLamlntR (SEQ
ID NO: 112), for a total of 10/ug per 10cm dish. Lipofectamine Plus reagent was used to transfect the cells according to the manufacturers protocol. Two days post-transfection zeocin was added to the medium at 500Ng/ml. The cells were maintained in selective medium until colonies formed. The colonies were then ring-cloned (see, e.g., McFarland, 2000, Methods Cell Sci, Mar;22(1):63-66).
C. Analysis of Clones (PCR, SEQUENCING).

Rectified Sheet (Rule 91) Genomic DNA was isolated from each of the candidate clones with the Wizard kit (Promega) and following the manufacturers protocol. The following primer set was used to analyze the genomic DNA isolated from the zeocin resistant clones: 5PacSV40 -CTGTTAATTAACTGTGGAATGTGTG TCAGTTAGGGTG (SEQ ID N0:76);
Antisense Zeo - TGAACAGGGTCACGTCGTCC (SEQ ID NO:77). PCR
amplification with the above primers and genomic DNA from the site-specific integration of any of the 4 zeocin vectors would result in a 673bp PCR product.

As set forth in Table 2, of the 4 zeocin resistant candidate clones thusfar analyzed by PCR, all 4 exhibit the correct sequence for a site-specific integration event.

Integration of a PCR product by site-specific recombination.
In this example a gene is integrated onto the platform ACes by site-specific recombination without cloning said gene into a vector.
A. PCR PRIMER DESIGN.
PCR primers are designed to contain an attB site at the 5' end of one of the primers in the primer set. The remaining primers, which could be one or more than one primer, do not contain an attB site, but are complementary to sequences flanking the gene or genes of interest and any associated regulatory sequences. In first example, 2 primers (one containing an attB site) are used to amplify a selective gene such as puromycin.
In a second example as shown in Figure 13, the primer set includes primers 1 & 2 that amplify the GFP gene without amplification of an upstream promoter. Primer 1 contains the attB site at the 5' end of the oligo. Primers 3 & 4 are designed to amplify the IRES-blasticidin DNA
sequences from the vector pIRESblasticidin. The 5'end of primer 3 contains sequences complementary to the 5' end of primer 2 such that annealing can occur between 5' ends of the two primers.
B. PCR REACTION AND SUBSEQUENT LIGATION TO CREATE
CIRCULAR MOLECULES FROM THE PCR PRODUCT
In the first example set forth above in Section A, the two PCR
primers are combined with a puromycin DNA template such as pPUR
(Clontech), a heat stable DNA polymerase and appropriate conditions for DNA amplification. The resulting PCR product (attB-Puromycin) is then then purified and self-ligated to form a circular molecule.
In the second example set forth above in Section A, amplification of the GFP gene and IRES-blasticidin sequences is accomplished by combining primers 1 & 2 with DNA template pD2eGFP and primers 3 & 4 with template piRESblasticidin under appropriate conditions to amplify the desired template. After initial amplification of the two products (attB-GFP
& IRES-blasticidin) in separate reactions, a second round of amplification using both of the PCR products from the first round of amplification together with primers 1 and 4 amplifies the fusion product attB-GFP-IRES-blasticidin (Figure 13). This technique of using complementary sequences in primer design to create a fusion product is employed in Saccharomyces cerevisiae for allele replacement (Erdeniz et a/ (1997) Gen Res 7:1174-1183). The amplified product is then purified from the PCR reaction mixture by standard methods and ligated to form a circular molecule.
C. INTRODUCTION OF PCR PRODUCT ONTO THE ACes USING A
RECOMBINASE
The circular PCR product is then be introduced to the platform ACes using the bacteriphage lambda integrase E174R. The introduction can be performed in vivo by transfecting the pCXLamlntR (SEQ ID NO:
112) vector encoding the lambda integrase mutant El 74R together with the circularized PCR product into a cell line containing the platform ACE.

D. SELECTION FOR MARKER GENE
The marker gene (in this case either puromycin, blasticidin or GFP) is used to enrich the population for cells containing the proper integration event. A proper integration event in the second example (Figure 14) juxtaposes a promoter residing on the platform ACes 5' to the attB-GFP-IRES-Blasticidin PCR product, allowing for transcription of both GFP and blasticidin. If enrichment is done by drug selection, blasticidin is added to the medium on the transfected cells 24-48 hours post-transfection.
Selection is maintained until colonies are formed on the plates. If enrichment is done by cell sorting, cells are sorted 2-4 days post-transfection to enrich for cells expressing the fluorescent marker (GFP in this case).
E. ANALYSIS OF CLONES
Clonal isolates are analyzed by PCR, FISH and sequence analysis to confirm proper integration events.

Construction of a human platform ACes "ACE 0.1 "
A. CONSTRUCTION OF THE TARGETING VECTOR pPACrDNA
Genome Systems (IncyteGenomics) was supplied with the primers 5'HETS (GGGCCGAAACGATCTCAACCTATT; SEQ ID NO:78), and 3'HETS (CGCAGCGGCCCTCCTACTC; SEQ ID NO:79), which were used to amplify a 538bp PCR product homologous to nt 9680-10218 of the human rDNA sequences (GenBank Accession No. U13369) and used as a probe to screen a human genomic P1 AC (P1 Artificial Chromosome) library constructed in the vector pCYPAC2 (loannou et aL (1994) Nat.
Genet. 6(1): 84-89). Genome Systems clone #18720 was isolated in this screen and contains three repeats of human rDNA as assessed by restriction analysis. GS clone #18720, was digested with Pmel, a restriction enzyme unique to a single repeat of the human rDNA (45Kbp), and then religated to form pPACrDNA (Figure 15). The insert in pPACrDNA was analyzed by restriction digests and sequence analysis of the 5' and 3' termini. The pPACrDNA, rDNA sequences are homologous to Genbank Accession #U13369, containing an insert of about 45 kB
comprising a single repeat beginning from the end of one repeat at -33980 (relative to the Genbank sequence) through the beginning of the next repeat up to approximately 35120 (the repeat offset from that listed in the GenBank file). Thus, the rDNA sequence is just over 1 copy of the repeat extending from 33980 (+/-10bp) to the end of the first repeat (43Kbp) and continuing into the second repeat to bp 35120 (+/-1Obp).
B. TRANSFECTION AND ACes FORMATION.
Five hundred thousand MSU1.1 cells (Morgan et al., 1991, Exp.
Cell Res., Nov;197(1):125-136; provided by Dr. Justin McCormick at Michigan State University) were plated per 6cm plate (3 plates total) and allowed to grow overnight. The cells were 70-80% confluent the following day. One plate was transfected with 15Ng pPACrDNA
(linearized with Pme I) and 2,ug pSV40attPsensePuro (linearized with Sca I; see Example 3). The remaining plates were controls and were transfected with either 20Ng pBS (Stratagene) or 20Ng pSV40attBsensePuro (linearized with Sca I). All three plates were transfected using a CaPO4 protocol.
C. SELECTION OF PUROMYCIN RESISTANT COLONIES
One day post-transfection the cells were "glycerol shocked" by the addition of PBS medium containing 10% glycerol for 30 seconds.
25. Subsequently, the glycerol was removed and replaced with fresh DMEM.
Four days post-transfection selective medium was added. Selective medium contains 1Ng/ml puromycin. The transfection plates were maintained at 37 C with 5% C02 in selective medium for 2 weeks at which point colonies could be seen on the plate transfected with Rectified Sheet (fluie 91) pPACrDNA and pSV40attPsensePuro. The colonies were ring-cloned from the plate on day 17 post-selection and expanded in selective medium for analysis. Only two colonies (M2-2d & M2-2b) were able to proliferate in the selective medium after cloning. No colonies were seen on the control plates after 37 days in selective medium.
D. ANALYSIS OF CLONES
FISH analysis was performed on the candidate clones to detect ACes formation. Metaphase spreads from the candidate clones were probed in multiple probe combinations. In one experiment, the probes used were biotin-labeled human alphoid DNA (pPACrDNA) and digoxigenin-labeled mouse major DNA (pFK161) as a negative control.
Candidate M2-2d was single cell subcloned by flow sorting and the candidate subclones were reanalyzed by FISH. Subclone 1131 of M2-2d was determined to be a platform ACes and is also designated human Platform ACE 0.1.

Site-specific integration of a marker gene onto a human platform ACE 0.1 The promoterless delivery method was used to deliver a promoterless blasticidin marker gene onto the human platform ACes with excellent results. The human ACes platform with a promoterless blasticidin marker gene resulted in 21 of 38 blasticidin resistant clones displaying a PCR product of the expected size from the population co-transfected with pLIT38attBBSRpoIyAlO and pCXLamIntR (Figure 8; SEQ
ID NOs. 111 and 112). Whereas, the population transfected with pBlueScript resulted in 0 blasticidin resistant colonies.
A. CONSTRUCTION OF pLIT38attB-BSRpolyA10 & pLIT38attB-BSRpolyA2.
The vector pLITMUS 38 (New England Biolabs; U.S. Patent No.
5,691,140; SEQ ID NO: 119) was digested with EcoRV and ligated to two annealed oligomers, which form an attB site (attB1 5'-TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA-3' (SEQ ID NO:8); attB2 5'- TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA-3'; SEQ ID NO:9).
This ligation reaction resulted in the vector pLlT38attB (SEQ ID NO: 120).
The blasticidin resistance gene and SV40 polyA site were PCR amplified with primers: 5BSD (ACCATGAAAACATTTAACATTTCTCAACA; SEQ ID
NO:80) and SV40polyA (TTTATTTGTGAAATTTGTGATGCTATTGC; SEQ
ID NO:81) using pPAC4 (Frengen, E., et al. (2000) Genomics 68 (2), 118-126; GenBank Accession No. U75992) as template. The blasticidin-SV40polyA PCR product was then ligated into pLIT38attB at the BamHI
site, which was Klenow treated following digestion with BamHl.
pLIT38attB-BSDpolyA10 (SEQ ID NO: 111) and pLIT38attB-BSDpolyA2 (SEQ ID NO: 121) are the two resulting orientations of the PCR product ligated into the vector.
B. TRANSFECTION OF MSU1.1 CELLS CONTAINING HUMAN
PLATFORM ACE 0.1.
MSU1.1 cells containing human platform ACE 0.1 (see Example 9) were expanded and plated to five 10cm dishes with 1.3x106 cells per dish. The cells were incubated overnight in DMEM with 10% fetal bovine serum, at 37 C and 5% CO2. The following day the cells were transfected with 5Ng of each plasmid as set forth in Table 3, for a total of 10Ng of DNA per plate of cells transfected (see Table 3) using ExGen 500 in vitro transfection reagent (MBI fermentas, cat. no. R051 1). The transfection was performed according to the manufacturers protocol.
Cells were incubated at 37 C with 5% CO2 in DMEM with 10% fetal bovine serum following the transfection.

Rectified Sheet (Rule 91) i Table 3 Plate # Plasmid 1 Plasmid 2 No. Bsd' Colonies 1 pBS None 0 2 pCXLamint pLIT38attB- 16 BSRpoIyA10 3 pCXLamintR pLIT38attB- 40 BSRpolyAl 0 4 pCXLamlnt pLIT38attB- 28 BSRpolyA2 5 pCXLamlntR pLIT38attB- 36 BSRpolyA2 C. SELECTION OF BLASTICIDIN RESISTANT CLONES.
Three days following the transfection the cells were split from a 10 cm dish to two 15cm dishes. The cells were maintained in DMEM with 10% fetal bovine serum for 4 days in the 15 cm dishes. Seven days post-transfection blasticidin was introduced into the medium. Stably transfected cells were selected with 1/.tg/ml blasticidin. The number of colonies formed on each plate is listed in Table 3. These colonies were ring-cloned and expanded for PCR analysis. Upon expansion in blasticidin containing medium some clones failed to live and therefore do not have corresponding PCR data.
D. PCR ANALYSIS
Thirty-eight of the 40 clones from plate 3 grew after ring-cloning.
Genomic DNA was isolated from these clones with the Promega Wizard Genomic cDNA purification kit, digested with EcoRl and used as template in a. PCR reaction with the following primers: 3BSP - TTAATTTCGGG
TATATTTGAGTGGA (SEQ ID NO:82); 5PacSV40 -CTGTTAATTAACTGTGGAA TGTGTGTCAGTTAGGGTG (SEQ ID NO:76).
The PCR conditions were as follows. 1 00ng of genomic DNA was amplified with 0.51j1 Herculase polymerase (Stratagene) in a 50u1 reaction that contained 12.5pmole of each primer, 2.5mM of each dNTP, and 1 X
Herculase buffer (Stratagene). The reactions were placed in a PerkinElmer thermocycler programmed as follows: Initial denaturation at 95 C for 10 minutes; 35 cycles of 94 C for 1 minute, 53 C for 1 minute, 72 C for 1 minute, and 72 C for 1 minute; Final extension for 10 minutes at 72 C;
and 4 C hold. If pLIT38attB-BSRpolyA10 integrates onto the human platform ACE 0.1 correctly, PCR amplification with the above primers should yield an 804bp product. Twenty-one of the 38 clones from plate 3 produced a PCR product of the expected 804bp size.

Delivery of a Vector comprising a Promoterless Marker Gene and a gene encoding a therapeutic product to a Platform ACes Platform ACes containing pSV40attPsensePURO (Figure 4) were constructed as set forth in Examples 3 and 4.
A. CONSTRUCTION OF DELIVERY VECTORS
1. Erythropoietin cDNA vector, p18EPOcDNA.
The erythropoietin cDNA was PCR amplified from a human cDNA
library (E. Perkins et al., 1999, Proc. Nat/. Acad. Sci. USA 96(5): 2204-2209) using the following primers: EPO5XBA -TATCTAGAATGGGGGTGC ACGAATGTCCTGCC (SEQ ID NO: 83);
EP03BSI - TACGTACGTCATC TGTCCCCTGTCCTGCAGGC (SEQ ID NO:
84). The cDNA was amplified through two successive rounds of PCR
using the following conditions: heat denaturation at 95 C for 3 minutes;
35 cycles of a 30 second denaturation (95 C), 30 seconds of annealing (60 C), and 1 minute extension (72 C); the last cycle is followed by a 7 minute extension at 72 C. BIO-X-ACT (BIOLINE) was used to amplify the erythropoietin cDNA from 2.5ng of the human cDNA library in the first round of amplification. Five p1 of the first amplification product was used Rectified Sheet (Fine 91) S

as template for the second round of amplification. Two PCR products were produced from the second amplification with Taq polymerase (Eppendorf), each product was cloned into pCR2.1-Topo (Invitrogen) and sequenced. The larger PCR product contained the expected cDNA
sequence for erythropoietin. The erythropoietin cDNA was moved from pTopoEPO into pl8attBZeo(6XHS4)2eGFP (SEQ ID NO: 110). pTopoEPO
was digested with BsiWl and Xbal to release a 588 bp EPO cDNA. BsrGl and BsiWl create compatable ends. The eGFP gene was removed from pl8attBZeo(6XHS4)2eGFP by digestion with BsiWI and Xbal, the 8.3 Kbp vector backbone was gel purified and ligated to the 588 bp EPO cDNA to create p18EPOcDNA (SEQ ID NO: 124).
2. Genomic erythropoietin vector, p18genEPO.
The erythropoietin genomic clone was PCR amplified from a human genomic library (Clontech) using the following primers: GENEP03BSI -CGTACGTCATCTGTCCCCT GTCCTGCA (SEQ ID NO: 85); GENEPO
5XBA -TCTAGAATGGGGGT GCACGGTGAGTACT (SEQ ID NO: 86). The reaction conditions for the amplification were as follows: heat denaturation for 3 minutes (95 C); 30 cycles of a 30 second denaturation (95 C), 30 seconds annealing (from 65 C decreasing 0.5 C per cycle to 50 C), and 3 minutes extension (72 C); 15 cycles of a 30 second denaturation (95 C), 30 seconds annealing (50 C), and 3 minute extension (72 C); the last cycle is followed by a 7 minute extension at 72 C. The erythropoietin genomic PCR product (2147 bp) was gel purified and cloned into pCR2.lTopo to create pTopogenEPO. Sequence analysis revealed 2bp substitutions and insertions in the intronic sequences of the genomic clone of erythropoietin. A partial digest with Xbal and complete digest with BsiWl excised the erythropoietin genomic insert from pTopogenEPO. The resulting 2158 bp genomic erythropoietin fragment was ligated into the 8.3 Kbp fragment resulting from the digestion of p 1 8attBZeo (6XHS4)2eGFP (SEQ ID NO: 110) with Xbal and BsrGI to create pl8genEPO (SEQ ID NO: 125).
B. TRANSFECTION AND SELECTION WITH DRUG

The erythropoietin genomic and cDNA genes were each moved onto the platform ACes B19-38 (constructed as set forth in Example 3) by co-transfecting with pCXLamIntR. Control transfections were also performed using pCXLamint (SEQ ID NO: 127) together with either p18EPOcDNA (SEQ ID NO: 124) or p18genEPO (SEQ ID NO: 125).
Lipofectamine Plus was used to transfect the DNA's into B19-38 cells according to the manufacturer's protocol. The cells were placed in selective medium (DMEM with 10% FBS and Zeocin @ 500ug/ml) 48 hours post-transfection and maintained in selective medium for 13 days.
Clones were isolated 15 days post-transfection.
C. ANALYSIS OF CLONES (ELISA, PCR) 1. ELISA Assays Thirty clones were tested for erythropoietin production by an ELISA
assay using a monoclonal anti-human erythropoietin antibody (R&D
Systems, Catalogue # MAB287), a polyclonal anti-human erythropoietin antibody (R & D Systems, Catalogue # AB-286-NA) and alkaline phosphotase conjugated goat-anti-rabbit IgG (heavy and light chains) (Jackson ImmunoResearch Laboratories, Inc., Catalogue # 111-055-144).
The negative control was a Zeocin resistant clone isolated from B19-38 cells transfected with pl8attBZeo(6XHS4) (SEQ ID NO: 117; no insert control vector) and pCXLamIntR (SEQ ID NO: 112). The preliminary ELISA assay was executed as follows: 1) Nunc-Immuno Plates (MaxiSorb 96-well, Catalogue # 439454) were coated with 75NI of a 1/200 dilution (in Phosphate buffered Saline, pH 7.4 (PBS), Sigma Catalogue # P-3813) of monoclonal anti-human erythropoietin antibody overnight at 4 C. 2) The following day the plates were washed 3 times with 300pl PBS

Rectified Sheet (Rule 91) containing 0.15% Tween 20 (Sigma, Catalogue # P-9416). 3) The plates were then blocked with :300111 of 1 % Bovine Serum Albumin (BSA; Sigma Catalogue # A-7030) in PBS for 1 hour at 37 C. 4) Repeat the washes as in step 2. 5) The clonal supernatants (75111 per clone per well of 96-well plate) were then added to the plate and incubated for 1 hour at 37 C.
The clonal supernatant analyzed in the ELISA assay had been maintained on the cells 7 days prior to analysis. .6) Repeat the washes of step 2. 7) Add 75pr1 of polyclonal anti-human erythropoietin antibody (1 /250 dilution in dilution buffer (0.5% BSA, 0.01 % Tween 20, 1 X PBS, pH 7.4) and incubate 1 hour at 37 C. 8) Repeat washes of step 2. 9) Add 75p1 of goat anti-rabbit conjugated alkaline, phosphatase diluted 1/4000 in dilution buffer and incubate 1 hour at 37 C. 10) Repeat washes. of step 2. 11) Add 75111 substrate, p-nitrophenyl phosphate (Sigma N2640), diluted to 1 mg/ml in substrate buffer (0.1 Ethanolamine-HCI (Sigma, Catalogue' # E-6133), 5mM MgCl2 (Sigma, Catalogue # M-2393), pH 9.8). incubate the plates in the dark for 1 hour at room temperature (22 C). 12) Read the absorption at 405nm (reference wavelength 495nm) on an Universal Microplate Reader (Bio-Tek Instruments, Inc., model # ELX800 UV). The erythropoietin standard curve was derived from readings of diluted human recombinant Erythropoietin (Roche, catalogue # 1-120-166; dilution range 125 - 7.8mUnits/ml). From this preliminary assay the 21 clones displaying the highest expression of erythropoietin were analyzed a second time in the same manner using medium supernatants that had, been on the clones for 24 hours and a 1:3 dilution therof.
2. PCR Analysis Genomic DNA was isolated from the 21 clones with the best expression (as assessed by the initial ELISA assay above) as well as the B19-38 cell line and used for PCR analysis. Genomic DNA was isolated using the Wizard genomic DNA purification kit (Promega) according to the *Trade-mark manufacturers protocol. Amplification was performed on 100ng of genomic DNA as template with MasterTaq DNA Polymerase (Eppendorf) and the primer set 5PacSV40 - CTGTTAATTAACTGTGGAATGTGTG
TCAGTTAGGGTG (SEQ ID NO: 76) and Antisense Zeo -TGAACAGGGTCACGTCGTCC (SEQ ID NO: 77). The amplification conditions were as follows: heat denaturation for 3 minutes (95 C); 30 cycles of a 30 second denaturation (95 C), 30 seconds annealing (from 65oC decreasing 0.5oC per cycle to 50 C), and 1 minutes extension (72 C); 15 cycles of a 30 second denaturation (95 C), 30 seconds annealing (50 C), and 1 minute extension (72 C); the last cycle is followed by a 10 minute extension at 72 C. PCR products were size separated by gel electrophoresis. Of the 21 clones analyzed 19 produced a PCR product of 650 bp as expected for a site-specific integration event.
All nineteen clones were the result of transformations with p 1 9EPOcDNA
(5) or p18genEPO (14) and pCXLamlntR (i.e. mutant integrase). The remaining two clones, both of which were the result of transformation with pl8genEPO (SEQ ID NO: 125) and pCXLamint (i.e. wildtype integrase; SEQ ID NO: 127), produced a 400 bp PCR product.

Preparation of a Transformation Vector Useful for the Induction of Plant Artificial Chromosome Formation Plant artificial chromosomes (PACs) can be generated by introducing nucleic acid, such as DNA, which can include a targeting DNA, for example rDNA or lambda DNA, into a plant cell, allowing the cell to grow, and then identifying from among the resulting cells those that include a chromosome with a structure that is distinct from that of any chromosome that existed in the cell prior to introduction of the nucleic acid. The structure of a PAC reflects amplification of chromosomal DNA, for example, segmented, repeat region-containing and heterochromatic Rectified pheet (Rul1e 91) = =

structures. It is also possible to select cells that contain structures that are precursors to PACs, for example, chromosomes containing more than one centromere and/or fragments thereof, and culture and/or manipulate them to ultimately generate a PAC within the cell.
In the method of generating PACs, the nucleic acid can be introduced into a variety of plant cells. The nucleic acid can include targeting DNA and/or a plant expressable DNA encoding one or multiple selectable markers (e.g., DNA encoding bialophos (bar) resistance) or scorable markers (e.g., DNA encoding GFP). Examples of targeting DNA
include, but are not limited to, N. tabacum rDNA intergenic spacer sequence (IGS) and Arabidopsis rDNA such as the 18S, 5.8S, 26S rDNA
and/or the intergenic spacer sequence. The DNA can be introduced using a variety of methods, including, but not limited to Agrobacterium-mediated methods, PEG-mediated DNA uptake and electroporation using, for example, standard procedures according to Hartmann et a/ [(1998) Plant Molecular Biology 36:7411. The cell into which such DNA is introduced can be grown under selective conditions and can initially be grown under non-selective conditions and then transferred to selective media. The cells or protoplasts can be placed on plates containing a selection agent to grow, for example, individual calli. Resistant calli can be scored for scorable marker expression. Metaphase spreads of resistant cultures can be prepared, and the metaphase chromosomes examined by FISH analysis using specific probes in order to detect amplification of regions of the chromosomes. Cells that have artificial chromosomes with functioning centromeres or artificial chromosomal intermediate structures, including, but not limited to, dicentric chromosomes, formerly dicentric chromosomes, minichromosomes, heterochromatin structures (e.g.
sausage chromosomes), and stable self-replicating artificial chromosomal intermediates as described herein, are Rectified Sheet (Rule 91) =05-56(S) identified and cultured. In particular, the cells containing self-replicating artificial chromosomes are identified.
The DNA introduced into a plant cell for the generation of PACs can be in any form, including in the form of a vector. An exemplary vector for use in methods of generating PACs can be prepared as follows.
For the production of artificial chromosomes, plant transformation vectors, as exemplified by pAgila and pAgllb, containing a selectable marker, a targeting sequence, and a scorable marker were constructed using procedures well known in the art to combine the various fragments.
The vectors can be prepared using vector pAg 1 as a base vector and inserting the following DNA fragments into pAgl: DNA encoding f-glucoronidase under the control of the nopaline synthase (NOS) promoter fragment and flanked at the 3' end by the NOS terminator fragment, a fragment of mouse satellite DNA and an N. tabacum rDNA intergenic spacer sequence (IGS). In constructing plant transformation vectors, vector pAg2 can also be used as the base vector.
1. Construction of pAG1 Vector pAg1 (SEQ. ID. NO: 89) is a derivative of the CAMBIA
vector named pCambia 3300 (Center for the Application of Molecular Biology to International Agriculture (CAMBIA), Canberra, Australia), which is a modified version of vector pCambia 1300 to which has been added DNA from the bar gene confering resistance to phosphinothricin. The nucleotide sequence of pCambia 3300 is provided in SEQ. ID. NO: 90. pCambia 3300 also contains a lacZ
alpha sequence containing a polylinker region.
pAg1 was constructed by inserting two new functional DNA
fragments into the polylinker of pCambia 3300: one sequence containing an attB site and a promoterless zeomycin resistance-encoding DNA
flanked at the 3' end by a SV40 polyA signal sequence, and a second =

sequence containing DNA from the hygromycin resistance gene (hygromycin phosphotransferase) confering resistance to hygromycin for selection in plants. Although the zeomycin-SV40 polyA signal fusion is not expected to function in plant cells, it can be activated in mammalian cells by insertion of a functional promoter element into the attB site by site-specific recombination catalyzed by the Lambda att integrase. Thus, the inclusion of the attB-zeomycin sequences allows for evaluation of functionality of plant artificial chromosomes in mammalian cells by activation of the zeomycin resistance-encoding DNA, and provides an att site for further insertion of new DNA sequences into plant artificial chromosomes formed as a result of using pAg1 for plant transformation.
The second functional DNA fragment allows for selection of plant cells with hygromycin. Thus, pAg1 contains DNA from the bar gene confering resisance to phosphinothricin, DNA from the hygromycin resistance gene, both resistance-encoding DNAs under the control of a separate cauliflower mosaic virus (CaMV) 35S promoter, and the attB-promoterless zeomycin resistance-encoding DNA.
pAg 1 is a binary vector containing Agrobacterium right and left T-DNA border sequences for use in Agrobacterium-mediated transformation of plant cells or protoplasts with the DNA located between the border sequences. pAg1 also contains the pBR322 Ori for replication in E.co/i.
pAg 1 was constructed by ligating Hindlll/Pstl-digested p3300attBZeo with Hindlll/Pstl-digested pBSCaMV35SHyg as follows.
a. Generation of p3300attBZeo Plasmid pCambia 3300 was digested with Pstl/Ec/136 II and ligated with Pstl/Stul-digested pLlTattBZeo (the nucleotide sequence of pLlTattBZeo is provided in SEQ. ID. NO: 91), which contains DNA
encoding the zeocin resistance gene and an attB Integrase recognition sequence, to generate p3300attBZeo which contains an attB site, a promoterless Rectified Sheet (Rule 91) . = =

zeomycin resistance-encoding DNA flanked at the 3' end by a SV40 polyA signal, and a reconstructed Pstl site.
b. Generation of pBSCaMV35SHyg A DNA fragment containing DNA encoding hygromycin phosphotransferase flanked by the CaMV 35S promoter and the CaMV
35S polyA signal sequence was obtained by PCR amplification of plasmid pCambia 1302 (GenBank Accession No. AF234298 and SEQ. ID. NO:
92). The primers used in the amplification reaction were as follows:
CaMV35SpoIyA:

5'-CTGAATTAACGCCGAATTAATTCGGGGGATCTG-3' SEQ. ID. NO: 93 CaMV35Spr:
5'-CTAGAGCAGCTTGCCAACATGGTGGAGCA-3' SEQ. ID. NO: 94 The 2100-bp PCR fragment was ligated with EcoRV-digested pBluescript II SK+ (Stratagene, La Jolla, CA, U.S.A.) to generate pBSCaMV35SHyg.
c. Generation of pAg1 To generate pAg1, pBSCaMV35SHyg was digested with Hindlll/Pstl and ligated with Hindlll/Psti-digested p3300attBZeo. Thus, pAg1 contains the pCambia 3300 backbone with DNA conferring resistance to phophinothricin and hygromycin under the control of separate CaMV 35S promoters, an attB-promoterless zeomycin resistance-encoding DNA recombination cassette and unique sites for adding additional markers, e.g., DNA encoding GFP. The attB site can be used as decribed herein for the addition of new DNA sequences to plant artificial chromosomes, including PACs formed as a result of using the pAg 1 vector, or derivatives thereof, in the production of PACs. The attB
site provides a convenient site for recombinase-mediated insertion of DNAs containing a homologous att site.
2. pAG2 Rectified Sheet (Rule 91) The vector pAg2 (SEQ. ID. NO: 95) is a derivative of vector pAg1 formed by adding DNA encoding a green fluorescent protein (GFP), under the control of a NOS promoter and flanked at the 3' end by a NOS polyA
signal, to pAg1. pAg2 was constructed as follows. A DNA fragment containing the NOS promoter was obtained by digestion of pGEM-T-NOS, or pGEMEasyNOS (SEQ. ID. NO: 96), containing the NOS promoter in the cloning vector pGEM-T-Easy (Promega Biotech, Madison, WI, U.S.A.), with Xbal/Ncol and was ligated to a Xbal/Ncol fragment of pCambia 1302 containing DNA encoding GFP (without the CaMV 35S promoter) to generate p1302NOS (SEQ. ID. NO: 97) containing GFP-encoding DNA in operable association with the NOS promoter. Plasmid p1302NOS was digested with Smal/BsiWl to yield a fragment containing the NOS
promoter and GFP-encoding DNA. The fragment was ligated with Pmel/Bs/1NI-digested pAg1 to generate pAg2. Thus, pAg2 contains DNA
from the bar gene confering resistance to phosphinothricin, DNA
conferring resistance to hygromycin, both resistance-encoding DNAs under the control of a cauliflower mosaic virus 35S promoter, DNA
encoding kanamycin resistance, a GFP gene under the control of a NOS
promoter and the attB-zeomycin resistance-encoding DNA. One of skill in the art will appreciate that other fragments can be used to generate the pAg1 and pAg2 derivatives and that other heterlogous DNA can be incorporated into pAgl and pAg2 derivatives using methods well known in the art.
3. pAglia and pAgllb transformation vectors Vectors pAglla and pAgllb were constructed by inserting the following DNA fragments into pAg1 : DNA encoding ,8-glucoronidase, the nopaline synthase terminator fragment, the nopaline synthase (NOS) promoter fragment, a fragment of mouse satellite DNA and an N. tabacum Rectified `hoce! (Rule91) S = .

rDNA intergenic spacer sequence (IGS). The construction of pAglla and pAgllb was as follows.
An N. tabacum rDNA intergenic spacer (IGS) sequence (SEQ. ID.
NO: 98; see also GenBank Accession No. YO8422; see also Borysyuk et al. (2000) Nature Biotechnology 18:1303-1306; Borysyuk et al. (1997) Plant Mol. Bio%35:655-660; U.S. Patent Nos. 6,100,092 and 6,355,860) was obtained by PCR amplification of tobacco genomic DNA. The IGS
can be used as a targeting sequence by virtue of its homology to tobacco rDNA genes; the sequence is also an amplification promoter sequence in plants. This fragment was amplified using standard PCR conditions (e.g., as described by Promega Biotech, Madison, WI, U.S.A.) from tobacco genomic DNA using the primers shown below:
NTIGS-Fl 5'- GTG CTA GCC AAT GTT TAA CAA GAT G- 3' (SEQ ID No. 99) and NTIGS-RI
5'-ATG TCT TAA AAA AAA AAA CCC AAG TGA C- 3' (SEQ ID No. 100) Following amplification, the fragment was cloned into pGEM-T Easy to give pIGS-I A fragment of mouse satellite DNA (Msatl fragment;
GenBank Accession No. V00846; and SEQ ID No. 101) was amplified via PCR from pSAT-1 using the following primers:

5'- AAT ACC GCG GAA GCT TGA CCT GGA ATA TCG C -3'(SEQ ID No.
102) and MSAT-Ri 5'-ATA ACC GCG GAG TCC TTC AGT GTG CA T- 3' (SEQ'ID No. 103) This amplification added a Sacll and a Hindlll site at the 5'end and a Sacll site at the 3' end of the PCR fragment. This fragment was then cloned into the Sacll site in pIGS-1 to give pMIGS-1, providing a eukaryotic RECTIFIED SHEET (RULE 91) centromere-specific DNA and a convenient DNA sequence for detection via FISH.
A functional marker gene containing a NOS-promoter:GUS:NOS
terminator fusion was then constructed containing the NOS promoter (GenBank Accession No. U09365; SEQ ID No. 104), E. coil ,Q-glucuronidase coding sequence (from the GUS gene; GenBank Accession No. S69414; and SEQ ID No. 105), and the nopaline synthase terminator sequence (GenBank Accession No. U09365; SEQ ID No. 107).
The NOS promoter in pGEM-T-NOS was added to a promoterless GUS
gene in pBlueScript (Stratagene, La Jolla, CA, U.S.A.) using NotI/Spel to form pNGN-1, which has the NOS promoter in the opposite orientation relative to the GUS gene.
pMIGS-1 was digested with Notl/Spel to yield a fragment containing the mouse major satellite DNA and the tobacco IGS which was then added to Noti-digested pNGN-1 to yield pNGN-2. The NOS promoter was then re-oriented to provide a functional GUS gene, yielding pNGN-3, by digestion and religation with Spel. Plasmid pNGN-3 was then digested with Hindlll, and the Hindlll fragment containing the $-glucuronidase coding sequence and the rDNA intergenic spacer, along with the Msat sequence, was added to pAG-1 to form pAglla (SEQ ID NO: 108), using the unique HindIll site in pAg 1 located near the right T-DNA border of pAg1, within the T-DNA region.
Another plasmid vector, referred to as pAgllb, was also recovered, which contained the inserted Hindill fragment (SEQ ID NO: 108) in the opposite orientation relative to that observed in pAglIa. Thus, pAglla and pAgllb differ only in the orientation of the Hindlll fragment containing the mouse major satellite sequence, the GUS DNA sequence and the IGS
sequence. The nucleotide sequence of pAglla is provided in SEQ. ID. NO:
109.

Rectified .0-4h eat (Rile 91) Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

'I

SEQUENCE LISTING
<110> CHROMOS MOLECULAR SYSTEMS, INC.
Perkins, Edward Perez, Carl Lindenbaum, Michael Greene, Amy Leung, Josephine Fleming, Elena Stewart, Sandra Shellard, Joan <120> CHROMOSOME-BASED PLATFORMS
<130> 24601-420PC

<140> Not Yet Assigned <141> Herewith <150> 60/294,758 <151> 2001-05-30 <150> 60/366,891 <151> 2002-03-21 <160> 129 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer: attPUP
<400> 1 ccttgcgcta atgctctgtt acagg 25 <210> 2 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Primer: attPDWN
<400> 2 cagaggcagg gagtgggaca aaattg 26 <210> 3 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> Primer: Lamint 1 <400> 3 ttcgaattca tgggaagaag gcgaagtcat gagcg 35 <210> '4 <211> 34 <212> DNA
<213> Artificial Sequence = =

<220>
<223> Primer: Lamint 2 <400> 4 ttcgaattct tatttgattt caattttgtc ccac 34 <210> 5 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 5 cggacaatgc ggttgtgcgt 20 <210> 6 <211> 46 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 6 cgcgcagcaa aatctagagt aaggagatca agacttacgg ctgacg 46 <210> 7 <211> 46 <212> DNA
<213> Artificial Sequence <220>
<223> Lambda INTER174rev <400> 7 cgtcagccgt aagtcttgat ctccttactc tagattttgc tgcgcg 46 <210> 8 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> attB]
<400> 8 tgaagcctgc ttttttatac taacttgagc gaa 33 <210> 9 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> attB2 <400> 9 ttcgctcaag ttagtataaa aaagcaggct tca 33 <210> 10 <211> 25 <212> DNA
<213> Artificial Sequence <220>

<223> Primer: attPdwn2 <400> 10 tcttctcggg cataagtcgg acacc 25 <210> 11 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:CMVen <400> 11 ctcacgggga tttccaagtc tccac 25 <210> 12 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:attPdwn <400> 12 cagaggcagg gagtgggaca aaattg 26 <210> 13 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:CMVEN2 <400> 13 caactccgcc ccattgacgc aaatg 25 <210> 14 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:L1 <400> 14 agtatcgccg aacgattagc tcttca 26 <210> 15 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:Fl rev <400> 15 gccgatttcg gcctattggt taaa 24 <210> 16 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:RED

<400> 16 ccgccgacat ccccgactac aagaa 25 <210> 17 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer:L2rev <400> 17 ttccttcgaa ggggatccgc ctacc 25 <210> 18 <211> 22118 <212> DNA
<213> Mus musculus <300>
<308> GenBank X82564 <309> 1996-04-09 <400> 18 gaattcccct atccctaatc cagattggtg gaataacttg gtatagatgt ttgtgcatta 60 aaaaccctgt aggatcttca ctctaggtca ctgttcagca ctggaacctg aattgtggcc 120 ctgagtgata ggtcctggga catatgcagt tctgcacaga cagacagaca gacagacaga 180 cagacagaca gacagacgtt acaaacaaac acgttgagcc gtgtgccaac acacacacaa 240 acaccactct ggccataatt attgaggacg ttgatttatt attctgtgtt tgtgagtctg 300 tctgtctgtc tgtctgtctg tctgtctgtc tatcaaacca aaagaaacca aacaattatg 360 cctgcctgcc tgcctgcctg cctacacaga gaaatgattt cttcaatcaa tctaaaacga 420 cctcctaagt ttgccttttt tctctttctt tatctttttc ttttttcttt tcttcttcct 480 tccttccttc cttccttcct tccttccttt ctttctttct ttctttcttt cttactttct 540 ttctttcctt cttacattta ttcttttcat acatagtttc ttagtgtaag catccctgac 600 tgtcttgaag acactttgta ggcctcaatc ctgtaagagc cttcctctgc ttttcaaatg 660 ctggcatgaa tgttgtacct cactatgacc agcttagtct tcaagtctga gttactggaa 720 aggagttcca agaagactgg ttatattttt catttattat tgcattttaa ttaaaattta 780 atttcaccaa aagaatttag actgaccaat tcagagtctg ccgtttaaaa gcataaggaa 840 aaagtaggag aaaaacttga ggctgtctgt ggatggtcga ggctgcttta gggagcctcg 900 tcaccattct gcacttgcaa accgggccac tagaacccgg tgaagggaga aaccaaagcg 960 acctggaaac aataggtcac atgaaggcca gccacctcca tcttgttgtg cgggagttca 1020 gttagcagac aagatggctg ccatgcacat gctgtctttc agcttggtga ggtcaaagta 1080 caaccgagtc acagaacaag gaagtataca cagtgagttc caggtcagcc agagtttaca 1140 cagagaaacc acatcttgaa aaaaacaaaa aaataaatta aataaatata atttaaaaat 1200 ttaaaaatag ccgggagtga tggcgcatgt ctttaatccc agctctcttc aggcagagat 1260 gggaggattt ctgagtttga ggccagcctg gtctgcaaag tgagttccag gacagtcagg 1320 gctatacaga gaaaccctgt cttgaaaact aaactaaatt aaactaaact aaactaaaaa 1380 aatataaaat aaaaatttta aagaatttta aaaaactaca gaaatcaaac ataagcccac 1440 gagatggcaa gtaactgcaa tcatagcaga aatattatac acacacacac acacagactc 1500 tgtcataaaa tccaatgtgc cttcatgatg atcaaatttc gatagtcagt aatactagaa 1560 gaatcatatg tctgaaaata aaagccagaa ccttttctgc ttttgttttc ttttgcccca 1620 agatagggtt tctctcagtg tatccctggc atccctgcct ggaacttcct ttgtaggttt 1680 ggtagcctca aactcagaga ggtcctctct gcctgcctgc ctgcctgcct gcctgcctgc 1740 ctgcctgcct gcctgcctca cttcttctgc cacccacaca accgagtcga acctaggatc 1800 tttatttctt tctctttctc tcttctttct ttctttcttt ctttctttct ttctttcttt 1860 ctttctttct ttcttattca attagttttc aatgtaagtg tgtgtttgtg ctctatctgc 1920 tgcctatagg cctgcttgcc aggagagggc aacagaacct aggagaaacc accatgcagc 1980 tcctgagaat aagtgaaaaa acaacaaaaa aaggaaattc taatcacata gaatgtagat 2040 atatgccgag gctgtcagag tgctttttaa ggcttagtgt aagtaatgaa aattgttgtg 2100 tgtcttttat ccaaacacag aagagaggtg gctcggcctg catgtctgtt gtctgcatgt 2160 agaccaggct ggccttgaac acattaatct gtctgcctct gcttccctaa tgctgcgatt 2220 aaaggcatgt gccaccactg cccggagtga tttcttcttt tttttttttt tggaaaatac 2280 ctttctttct ttttctctct ctctttcttc cttccttcct ttctttctat tctttttttc 2340 tttctttttt cttttttttt ttttttttaa aatttgccta aggttaaagg tgtgctccac 2400 aattgcctca gctctgctct aattctcttt aaaaaaaaac aaacaaaaaa aaaaccaaaa 2460 cagtatgtat gtatgtatat ttagaagaaa tactaatcca ttaataactc ttttttccta 2520 aaattcatgt cattcttgtt ccacaaagtg agttccagga cttaccagag aaaccctgtg 2580 ttcaaatttc tgtgttcaag gtcaccctgg cttacaaagt gagttccaag tccgataggg 2640 ctacacagaa aaaccatatc tcagaaaaaa aaaaagttcc aaacacacac acacacacac 2700 acacacacac acacacacac acacacacac acacacacag cgcgccgcgg cgatgagggg 2760 aagtcgtgcc taaaataaat atttttctgg ccaaagtgaa agcaaatcac tatgaagagg 2820 tactcctaga aaaaataaat acaaacgggc tttttaatca ttccagcact gttttaattt 2880 aactctgaat ttagtcttgg aaaagggggc gggtgtgggt gagtgagggc gagcgagcag 2940 atgggcgggc gggcgggtga gtgcccgtgg gcggtggcag cgagcaccag aaaacaacaa 3000 accccaagcg gtagagtgtt ttaaaaatga gacctaaatg tggtggaacg gaggtcgccg 3060 ccaccctcct cttccactgc ttagatgctc ccttcccctt actgtgctcc cttcccctaa 3120 ctgtgcctaa ctgtgcctat tccctcaccc cgctgattcg ccagcgacgt actttgactt 3180 caagaacgat tttgcctgtt ttcaccgctc cctgtcatac tttcgttttt gggtgcccga 3240 gtctagcccg ttcgctatgt tcgggcggga cgatggggac cgtttgtgcc actcgggaga 3300 agtggtgggt gggtacgctg ctccgtcgtg cgtgcgtgag tgccggaacc tgagctcggg 3360 agaccctccg gagagacaga atgagtgagt gaatgtggcg gcgcgtgacg gatctgtatt 3420 ggtttgtatg gttgatcgag accattgtcg ggcgacacct agtggtgaca agtttcggga 3480 acgctccagg cctctcaggt tggtgacaca ggagagggaa gtgcctgtgg taagtcgacc 3540 agggtgacag gagcccgggc aagcaggcgg gagcgtctcg gagatggtgt cgtgtttaag 3600 gacggtctct aacaaggagg tcgtacaggg agatggccaa agcagaccga gttgctgtac 3660 gcccttttgg gaaaaatgct agggttggtg gcaacgttac taggtcgacc agaaggctta 3720 agtcctaccc ccccccccct tttttttttt tttcctccag aagccctctc ttgtccccgt 3780 caccgggggc accgtacatc tgaggccgag aggacgcgat gggcccggct tccaagccgg 3840 tgtggctcgg ccagctggcg cttcgggtct tttttttttt tttttttttt ttttcctcca 3900 gaagccttgt ctgtcgctgt caccgggggc gctgtacttc tgaggccgag aggacgcgat 3960 gggccccggc ttccaagccg gtgtggctcg gccagctgga gcttcgggtc tttttttttt 4020 tttttttttt tttttttctc cagaagcctt gtctgtcgct gtcaccgggg gcgctgtact 4080 tctgaggccg agaggacgcg atgggtcggc ttccaagccg atgtggcggg gccagctgga 4140 gcttcgggtt tttttttttc ctccagaagc cctctcttgt ccccgtcacc gggggcgctg 4200 tacttctgag gccgagagga cgtgatgggc ccgggttcca ggcggatgtc gcccggtcag 4260 ctggagcttt ggatcttttt tttttttttt cctccagaag ccctctcttg tccccgtcac 4320 cgggggcacc ttacatctga gggcgagagg acgtgatggg tccggcttcc aagccgatgt 4380 ggcggggcca gctggagctt cgggtttttt ttttttcctc cagaagccct ctcttgtccc 4440 cgtcaccggg ggcgctgtac ttctgaggcc gagaggacgt gatgggcccg ggttccaggc 4500 ggatgtcgcc cggtcagctg gagctttgga tcattttttt ttttccctcc agaagccctc 4560 tcttgtcccc gtcaccgggg gcaccgtaca tctgaggccg agaggacacg atgggcctgt 4620 cttccaagcc gatgtgcccc ggccagctgg agcttcgggt cttttttttt ttttttcctc 4680 cagaagcctt gtctgtcgct gtcacccggg gcgctgtact tctgaggccg agaggacgcg 4740 atgggcccgg cttccaagcc ggtgtggctc ggccagctgg agcttcgggt cttttttttt 4800 tttttttttt ttcctccaga aaccttgtct gtcgctgtca cccggggcgc ttgtacttct 4860 gatgccgaga ggacgcgatg ggcccgtctt ccaggccgat gtggcccggt cagctggagc 4920 tttggatctt tttttttttt ttttcctcca gaagccctct cttgtccccg tcaccggggg 4980 caccttacat ctgaggccta gaggacacga tgggcccggg ttccaggccg atgtggcccg 5040 gtcagctgga gctttggatc tttttttttt ttttcttcca gaagccctct tgtccccgtc 5100 accggtggca ctgtacatct gaggcggaga ggacattatg ggcccggctt ccaatccgat 5160 gtggcccggt cagctggagc tttggatctt attttttttt taattttttc ttccagaagc 5220 cctcttgtcc ctgtcaccgg tggcacggta catctgaggc cgagaggaca ttatgggccc 5280 ggcttccagg ccgatgtggc ccggtcagct ggagctttgg atcttttttt ttttttttct 5340 tttttcctcc agaagccctc tctgtccctg tcaccggggg ccctgtacgt ctgaggccga 5400 gggaaagcta tgggcgcggt tttctttcat tgacctgtcg gtcttatcag ttctccgggt 5460 tgtcagggtc gaccagttgt tcctttgagg tccggttctt ttcgttatgg ggtcattttt 5520 gggccacctc cccagatacg acttccaggc gtcgttgctc gcctgtcact ttcctccatg 5580 tctcttttat gcttgtgatc ttttctatct gttcctattg gacctggaga taggtactga 5640 cacgctgtcc tttccctatt aacactaaag gacactataa agagaccctt tcgatttaag 5700 gctgttttgc ttgtccagcc tattcttttt actggcttgg gtctgtcgcg gtgcctgaag 5760 ctgtccccga gccacgcttc ctgctttccc gggcttgctg cttgcgtgtg cttgctgtgg 5820 gcagcttgtg acaactgggc gctgtgactt tgctgcgtgt cagacgtttt tcccgatttc 5880 cccgaggtgt cgttgtcaca cctgtcccgg ttggaatggt ggagccagct gtggttgagg 5940 gccaccttat ttcggctcac tttttttttt tttttttctc ttggagtccc gaacctccgc 6000 tcttttctct tcccggtctt tcttccacat gcctcccgag tgcatttctt tttgtttttt 6060 ttcttttttt tttttttttt ttggggaggt ggagagtccc gagtacttca ctcctgtctg 6120 tggtgtccaa gtgttcatgc cacgtgcctc ccgagtgcac ttttttttgt ggcagtcgct 6180 cgttgtgttc tcttgttctg tgtctgcccg tatcagtaac tgtcttgccc cgcgtgtaag 6240 acattcctat ctcgcttgtt tctcccgatt gcgcgtcgtt gctcactctt agatcgatgt 6300 ggtgctccgg agttctcttc gggccagggc caagccgcgc caggcgaggg acggacattc 6360 atggcgaatg gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420 cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480 cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540 tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600 = s ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660 gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720 gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780 tgtctgagaa gcccgttaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840 tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900 ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960 gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020 aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080 ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcgttg gccggggctc 7140 gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200 ggaaatggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc Ccctggaggg 7260 ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320 gggctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380 ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440 gcacaccccc gcgtgggcgt actttcctcc cctcctgagg gccgccgtgc ggccggggtg 7500 tgggtaggcg acggtgggct ccctggtcgc cacccgtctt ccctgtcctc acccgtgcct 7560 tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620 ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680 ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740 cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800 gttctgtg g agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860 gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920 cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980 ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040 tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100 gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160 ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220 acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280 cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340 gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400 gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460 agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520 ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580 accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640 gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700 cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760 catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820 tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880 ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940 gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000 gagcccctgc cgcacccgcc ggtgtgcggt ttcgcctcgc ggtcagttgg gccctggcgt 9060 tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120 gacgggtggc ctatccaggg ctcgcccccg ccgacctccg cctgcccgtc ccggtggtgg 9180 tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240 tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300 cgtcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360 gggacggagg ggagagcggg taagagaggt gtcggacagc tgtcccgggg cgacgctcgg 9420 gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480 tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540 cctcccgcga ggctctccgc cgccgccgcc tccttctcct ctctcgcgct ctctgtcccg 9600 ccttgtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660 ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720 agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780 ctacttggat aactgtggta attctagagc taatacatgc cgacggccgc tgacccccct 9840 tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900 ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960 acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020 tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080 gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140 cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200 ggaatgagtc cactttaaat cctttaacga ggatcaagtg gagggcaagt ctggtgccag 10260 cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320 tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380 ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440 gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctg a taccgcagct 10500 agtaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560 taagagggac ggCCgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620 = =

gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680 tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740 cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800 ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860 ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920 cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980 ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040 ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100 gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160 tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220 aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280 gaattcccag taagtgcggg tcataagctt gcgttaatca agtccctgcc ctttgtacac 11340 accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400 ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460 agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520 ctgtggagga gcggcggcgt ggcccgctct cccggtcttg tgtgtgtcct cgccgggagg 11580 cacgtgcctc cccggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640 gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cccccgattt cccctccccc 11700 tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760 gcgtctttcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820 ggtttttgac ccgtccggag ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880 cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940 ggatgtgagt gtcgtgtctg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000 gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060 ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120 cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180 cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240 cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300 gggttcCcgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360 cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420 cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480 cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540 tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600 catcgacact tcgaacgcac ttgcggcccc gggttcctcc cgtgtctacg cctgtctgag 12660 cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720 ctcgcagggc caacccccca acgcgggtcg ggccctccgt cccgcgaagt tcagacgtgt 12780 gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840 cgccctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900 gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960 tgggaaccca ccgcgccccc gtggcgcccg ggggtggggg cgtccgcatc tgctctggtc 13020 gaggttggcg gttgagggtg tgcgtgcgcc ggggtggtgg tcggtcccct gcggccgcgg 13080 ggttctcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140 gtttgggtct tgcgttgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200 ggcgccgcgc accgtccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260 gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320 cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380 cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440 ccgcgggacg ccgcggcttc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500 gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560 ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccaccCc cgccgccctc 13620 ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680 gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740 tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800 attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860 gaagagccca gcgccgaatc cccgccgggc gtcgcggcgt gggaaatgtg gcgtacggaa 13920 gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980 tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcggatcttc ccggagtcgg 14040 gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100 cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160 tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag '14220 gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280 ccccgttcct cccgacccct ccacccgccc gtcgttcccc tcttcctccc cgcgtccggc 14340 gcctccggcg gcggccgcgg ggggtggtgt gctggtgggg cgcgggcggg gccgggggtg 14400 gggtcggcgg gggaCcgccc ccagccggcg accggccgcc gccgggcgca cttccaccgt 14460 ggcggtgcgc cgcgaccggc tccgggacgg ccgggaagcc ccggtgggga aggtggctcg 14520 gggtgggcgg cgcgtctcag ggcgcgctga accacctcac cccgagtgtt acagccctcc 14580 ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640 = = ' ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg gcccggtccg cccctcccac 14700 -ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg-gggggcgggg----14760 cggactgtcc ccagtgcgcc cgggcggtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820 gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880 cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940 gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000 cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060 aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgagcaggg 15120 cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180 cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240 tttccctcag gatagctggc gctctcgctc cgagcgtacg cagttttatc cggtaaagcg 15300 aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360 agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420 ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480 gacgctcatc agaccccaga aaaggtgttg gtagatttag acagcaggac ggtggccatg 15540 gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600 aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660 ggacgggagc ggccgcgggt gcgcgtctct cggggtcggg ggtgcgtggc gggggcccgt 15720 cccccgcctc ccctccgcgc gccgggttcg cccccgcggc gtcgggcccc gcggagccta 15780 cgccgcgacg agtaggaggg ccgctgcggt gagccttgaa gcctagggcg cgggcccggg 15840 tggagccgcc gcaggtgcag atcttggtgg tagtagcaaa tattcaaacg agaactttga 15900 aggccgaagt ggagaagggt tccatgtgaa cagcagttga acatgggtca gtcggtcctg 15960 agagatgggc gagtgccgtt cgaaagggac gggcgatggc ctccgttgcc ctcggccgat 16020 cgaaagggag tcgggttcag atccccgaat CCggagtggc ggagatgggc gccgcgaggc 16080 cagtgcggta acgcgaccga tcccggagaa gccggcggga ggcctcgggg agagttctct 16140 tttctttgtg aagggcaggg cgccctggaa tgggttcgcc ccgagagagg ggcccgtgcc 16200 ttggaaagcg tcgcggttcc gggggcgtcc ggtgagctct cgctgcccct tgaaaatccg 16260 ggggagaggg tgtaaatctc gcgccgggcc gtacccatat ccgcagcagg tctccaaggt 16320 gaacagcctc tggcatgttg gaacaatgta ggtaagggaa gtcggcaagc cggatccgta 16380 acttcgggat aaggattggc tctaagggct gggtcggtcg ggctggggcg cgaagcgggg 16440 ctgggcgcgc gccgcggctg gacgaggcgc cgccgccctc tcccacgtcc ggggagaccc 16500 cccgtccttt ccgcccgggc ccgccctccc ctcttccccg cggggccccg tcgtcccccg 16560 cgtcgtcgcc acctctcttc ccccctcctt cttcccgtcg gggggcgggt cgggggtcgg 16620 cgcgcggcgc gggctccggg gcggcgggtc caaccccgcg ggggttccgg agggggagga 16680 accagcggtc cccggtgggg cggggggccc ggacactcgg ggggccggcg gcggcggcga 16740 ctctggacgc gagccgggcc cttcccgtgg atcgcctcag ctgcggcggg cgtcgcggcc 16800 ggtctcgggg accgcggcgg gtgccggcgc gggtcccctc cccgcggggc ctccctccac 16860 ccccccatcg cctctcccga ggtgcgtggc gggggcgggc gggcgtgtcc cgcgcgtgtg 16920 gggggaacct ccgcgtcggt gttcccccgc cgggttcggC ccccgggccg cggttttccg 16980 cgcggcgccc ccgcctgggc cggcgcctag cagccgactt agaactggtg cggaccaggg 17040 gaatccgact gtttaattaa aacaaagcat cgcgaaggcc cgcggcgggt gttgacgcga 17100 tgtgatttct gcccagtgct ctgaatgtca aagtgaagaa attcaatgaa gcgcgggtaa 17160 agggcgggag taactatgac tctcttaagg tagccaaatg cctcgtgatc taattagtga 17220 cgcgcatgaa tggatgaacg agattcccac tgtccctacc tactatccag cgaaaccaca 17280 gccaagggaa cgggcttggc ggaatcagcg gggaaagaag accctgttga gcttgactct 17340 agtctggcac ggtgaagaga catgagaggt gtagaataag tgggaggccc ccggcgcccg 17400 gccccgtcct cgcgtcgggg tcgggtcacg ccggcctcgc gggccgccgg tgaaatacca 17460 ctactctcat cgttttttca ctgacccggt gaggcggggg ggcgagcccc gaggggctct 17520 cgcttctggc gccaagcgtc cgtcccgcgc gtgcggccgg gcgcgacccg ctccggggac 17580 agtgccaggt ggggagtttg actggggcgg tacacctgtc aaacggtaac gcaggtgtcc 17640 taaggcgagc tcagggagga cagaaacctc ccgtggagca gaagggcaaa agctcgcttg 17700 atcttgattt tcagtacgaa tacagaccgt gaaagcgggg cctcacgatc cttctgacct 17760 tttgggtttt aagcaggagg tgtcagaaaa gttaccacag ggataactgg cttgtggcgg 17820 ccaagcgttc atagcgacgt cgctttttga tccttcgatg tcggctcttc ctatcattgt 17880 gaagcagaat tcaccaagcg ttggattgtt cacccactaa tagggaacgt gagctgggtt 17940 tagaccgtcg tgagacaggt tagttttacc ctactgatga tgtgttgttg ccatggtaat 18000 cctgctcagt acgagaggaa ccgcaggttc agacatttgg tgtatgtgct tggctgagga 18060 gccaatgggg cgaagctacc atctgtggga ttatgactga acgcctctaa gtcagaatcc 18120 gcccaagcgg aacgatacgg cagcgccgaa ggagcctcgg ttggccccgg atagccgggt 18180 ccccgtccgt cccgctggcc ggggtccccg cgtcgccccg cggcggcgcg gggtctcccc 18240 ccgCCgggcg tcgggaccgg ggtccggtgc ggagagccgt tcgtcttggg aaacggggtg 18300 cggccggaaa gggggccgcc ctcttgcccg tcacgttgaa cgcacgttcg tgtggaacct 18360 ggcgctaaac cattcgtaga cgacctgctt ctgggtcggg gtttcgtacg tagcagagca' 18420 gctccctcgc tgcgatctat tgaaagtcag ccctcgacac aagggtttgt ctctgcgggc 18480 tttcccgtcg cacgcccgct cgctcgcacg cgaccgtgtc gccgcccggg cgtaacgggg 18540 gcggtcgcct cggcccccgc gcggttgccc gaacgaccgt gtggtggttg ggggggggat 18600 cgtcttctcc tccgtctccc gaggacggtt cgtttctctt tccccttccg tcgctctcct 18660 tgggtgtggg agcctcgtgc cgtcgcgacc gcggcctgcc gtcgcctgcc gccgcagccc 18720 cttgccctcc ggccttggcc aagccggagg gcggaggagg gggatcggcg gcggcggcga 18780 ccgcggcgcg gtgacgcacg gtgggatccc catcctcggc gCgtccgtcg gggacggccg 18840 gttggagggg cgggaggggt ttttcccgtg aacgccgcgt tcggcgccag gcctctggcg 18900 gccggggggg cgctctctcc gcccgagcat ccccactccc gcccctcctc ttcgcgcgcc 18960 gcggcggcga cgtgcgtacg aggggaggat gtcgcggtgt ggaggcggag agggtccggc 19020 gcggcgcctc ttccattttt tcccccccaa cttcggaggt cgaccagtac tccgggcgac 19080 actttgtttt ttttttttcc cccgatgctg gaggtcgacc agatgtccga aagtgtcccc 19140 cccccccccc ccccccggcg cggagcggcg gggccactct ggactctttt tttttttttt 19200 tttttttttt ttaaattcct ggaaccttta ggtcgaccag ttgtccgtct tttactcctt 19260 catataggtc gaccagtaat ccgggtggta ctttgtcttt ttctgaaaat cccagaggtc 19320 gaccagatat ccgaaagtcc tctctttccc tttactcttc cccacagcaa ttctcttttt 19380 tttttttttt tttggtgtgc ctctttttga cttatataca tgtaaatagt gtgtacgttt 19440 atatacttat aggaggaggt cgaccagtac tccgggcgac actttgtttt tttttttttt 19500 tccaccgatg atggaggtcg accagatgtc cgaaagtgtc ccgtcccccc cctccccccc 19560 ccgcgacgcg gcgggctcac tctggactct tttttttttt tttttttttt tttaaatttc 19620 tggaacctta aggtcgacca gttgtccgtc tttcactcat tcatataggt cgaccggtgg 19680 tactttgtct ttttctgaaa atcgcagagg tcgaccagat gtcagaaagt ctggtggtcg 19740 ataaattatc tgatctagat ttgtttttct gtttttcagt tttgtgttgt tttgtgttgt 19800 tttgtgttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 19860 tttgtgttgt gttgtgttgt gttgtgttgg gttgggttgg gttgggttgg gttgggttgg 19920 gttgggttgg gttgggttgt gttgtttggt tttgtgttgt ttggtgttgt tggttttgtt 19980 ttgtttgctg ttgttttgtg ttttgcgggt cgaacagttg tccctaaccg agtttttttg 20040 tacacaaaca tgcacttttt ttaaaataaa tttttaaaat aaatgcgaaa atcgaccaat 20100 tatccctttc cttctctctc ttttttaaaa attttctttg tgtgtgtgtg tgtgtgtgtg 20160 tgtgtgtgtg tgcgtgtgtg tgtgtgtgtg cgtgcagcgt gcgcgcgctc gttttataaa 20220 tacttataat aataggtcgc cgggtggtgg tagcttcccg gactccagag gcagaggcag 20280 gcagacttct gagttcgagg ccagcctggt ctacagagga accctgtctc gaaaaatgaa 20340 aataaataca tacatacata catacataca tacatacata catacataca tacatatgag 20400 gttgaccagt tgtcaatcct ttagaatttt gtttttaatt aatgtgatag agagatagat 20460 aatagataga tggatagagt gatacaaata taggtttttt tttcagtaaa tatgaggttg 20520 attaaccact tttccctttt taggtttttt tttttttccc ctgtccatgt ggttgctggg 20580 atttgaactc aggaccctgg caggtcaact ggaaaacgtg ttttctatat atataaatag 20640 tggtctgtct gctgtttgtt tgtttgcttg cttgcttgct tgcttgcttg cttgcttgct 20700 tgcttttttt tttcttctga gacagtattt ctctgtgtaa cctggtgccc tgaaactcac 20760 tctgtagacc agcctggcct caatcgaact cagaaatcct cctgcctctt gtctacctcc 20820 caattttgga gtaaaggtgt gctacaccac tgcctggcat tattatcatt atcattatta 20880 attttattat tagacagaac gaaatcaact agttggtcct gtttcgttaa ttcatttgaa 20940 attagttgga ccaattagtt ggctggtttg ggaggtttct tttgtttccg atttgggtgt 21000 ttgtggggct ggggatcagg tatctcaacg gaatgcatga aggttaaggt gagatggctc 21060 gatttttgta aagattactt ttcttagtct gaggaaaaaa taaaataata ttgggctacg 21120 tttcattgct tcatttctat ttctctttct ttctttcttt ctttcagata aggaggtcgg 21180 ccagttcctc ctgccttctg gaagatgtag gcattgcatt gggaaaagca ttgtttgaga 21240 gatgtgctag tgaaccagag agtttggatg tcaagccgta taatgtttat tacaatatag 21300 aaaagttcta acaaagtgat ctttaacttt tttttttttt tttctccttc tacttctact 21360 tgttctcact ctgccaccaa cgcgctttgt acattgaatg tgagctttgt tttgcttaac 21420 agacatatat tttttctttt ggttttgctt gacatggttt ccctttctat ccgtgcaggg 21480 ttcccagacg gccttttgag aataaaatgg gaggccagaa ccaaagtctt ttgaataaag 21540 caccacaact ctaacctgtt tggctgtttt ccttcccaag gcacagatct ttcccagcat 21600 ggaaaagcat gtagcagttg taggacacac tagacgagag caccagatct cattgtgggt 21660 ggttgtgaac cacccaccat gtggttgcct gggatttgaa ctcaggatct tcagaagacg 21720 agtcagggct ct'aaaccgat gagccatctc tccagccctc ctacattcct tcttaaggca 21780 tgaatgatcc cagcatggga agacagtctg ccctctttgt ggtatatcac catatactca 21840 ataaaataat gaaatgaacg aagtctccac gtatttattt ctttcagata tctaaattct 21900 ctcacagcac cttcccctcc cccacactgc ctttctccct atgtttgggt ggggctgggg 21960 gaggggtggg gtgggggcag ggatctgcat gtcttcttgc aggtctgtga actatttgcg 22020 atggcctggt tctctgaact gttgagcctt gtctatccag aggctgactg gctagttttc 22080 tacctgaagt ccctgagtga tgatttccct gtgaattc 22118 <210> 19 <211> 175 <212> DNA
<213> Mus musculus <400> 19 ctcccgcgcg gcccccgtgt tcgccgttcc cgtggcgcgg acaatgcggt tgtgcgtcca 60 cgtgtgcgtg tccgtgcagt gccgttgtgg agtgcctcgc tctcctcctc ctccccggca 120 =

gcgttcccac ggttggggac caccggtgac ctcgccctct tcgggcctgg atccg 175 <210> 20 <211> 755 <212> DNA
<213> Mus musculus <400> 20 ggtctggtgg gaattgttga cctcgctctc gggtgcggcc tttggggaac ggcggggtcg 60 gtcgtgcccg gcgccggacg tgtgtcgggg cccacttccc gctcgagggt ggcggtggcg 120 gcggcgttgg tagtctcccg tgttgcgtct tcccgggctc ttgggggggg tgccgtcgtt 180 ttgggggccg gctttgcttg gcttacgcag gcttggtttg ggactgcctc aggagtcgtg 240 ggcggtgtga ttcccgccgg ttttgcctcg cgtctgcctg ctttgcctcg ggtttgcttg 300 gttcgtgtct cgggagcggt ggtttttttt tttttcgggt cccggggaga ggggtttttc 360 cgggggacgt tcccgtcgcc ccctgccgcc ggtgggtttt cgtttcgggc tgtgttcgtt 420 tccccttccc cgtttcgccg tcggttctcc ccggtcggtc ggccctctcc ccggtcggtc 480 gcccggccgt gctgccggac ccccccttct gggggggatg cccgggcacg cacgcgtccg 540 ggcggccact gtggtccggg agctgctcgg caggcgggtg agccagttgg aggggcgtca 600 tgcccccgcg ggctcccgtg gccgacgcgg cgtgttcttt gggggggcct gtgcgtgcgg 660 gaaggctgcg cacgttgtcg gtccttgcga gggaaagagg cttttttttt ttagggggtc 720 gtccttcgtc gtcccgtcgg cggtggatcc ggcct 755 <210> 21 <211> 463 <212> DNA
<213> Mus musculus <400> 21 ggccgaggtg cgtctgcggg ttggggctcg tccggccccg tcgtcctccg ggaaggcgtt 60 tagcgggtac cgtcgccgcg ccgaggtggg cgcacgtcgg tgagataacc ccgagcgtgt 120 ttctggttgt tggcggcggg ggctccggtc gatgtcttcc cctccccctc tccccgaggc 180 caggtcagcc tccgcctgtg ggcttcgtcg gccgtctccc=cccccctcac gtccctcgcg 240 atcgagcccg tccgttcgac cttccttccg ccttcccccc atctttccgc gctccgttgg 300 ccccggggtt ttcacggcgc cccccacgct cctccgcctc tccgcccgtg gtttggacgc 360 ctggttccgg tctccccgcc aaaccccggt tgggttggtc tccggccccg gcttgctctt 420 cgggtctccc aacccccggc cggaagggtt cgggggttcc ggg 463 <210> 22 <211> 378 <212> DNA
<213> Mus musculus <400> 22 ggattcttca ggattgaaac ccaaaccggt tcagtttcct ttccggctcc ggccgggggg 60 ggcggccccg ggcggtttgg tgagttagat aacctcgggc cgatcgcacg ccccccgtgg 120 cggcgacgac ccattcgaac gtctgcccta tcaactttcg atggtagtcg atgtgcctac 180 catggtgacc acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 240 ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga cccggggagg 300 tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtccac 360 tttaaatcct ttaagcag 378 <210> 23 <211> 378 <212> DNA
<213> Mus musculus <400> 23 gatccattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 60 tattaaagtt gctgcagtta aaaagctcgt agttggatct tgggagcggg cgggggttcc 120 gccgcgaggc gagtcaccgc ccgtccccgc cccttgcctc tcggcgcccc ctcgatgctc 180.
ttagctgagt tgtcccgcgg ggcccgaagc gtttactttg aaaaaattag agttgtttca 240 aagcaggccc gagccgcctg gataccgcca gctaggaaat aatggaatag gaccgcggtt 300 cctattttgt ttggttttcg gaactgagcc catgattaag ggaaacggcc gggggcattc 360 ccttattgcg ccccccta 378 <210> 24 <211> 719 <212> DNA
<213> Mus musculus <400> 24 ggatctttcc cgctccccgt tcctcccggc ccctccaccc gcgcgtctcc ccccttcttt 60 tcccctctcc ggaggggggg gaggtggggg cgcgtgggcg gggtcggggg tggggtcggc 120 gggggaccgc ccccggccgg caaaaggccg ccgccgggcg cacttcaacc gtagcggtgc 180 gccgcgaccg gctacgagac ggctgggaag gcccgacggg gaatgtggct cggggggggc 240 ggcgcgtctc agggcgcgcc gaaccacctc accccgagtg ttacagccct ccggccgcgc 300 tttcgcggaa tcccggggcc gaggggaagc ccgatacccg tcgccgcgct tttcccctcc 360 ccccgtccgc ctcccgggcg ggcgtggggg tgggggccgg gccgcccctc ccacccccgt 420 ggtttctctc tctcccggtc tcggccggtt tggggggggg agcccggttg ggggcggggc 480 ggactgtcct cagtgcgccc cgggcgtcgt cgcgccgtcg ggcccggggg gttctctcgg 540 tcacgccgcc cccgacgaag ccgagcgcac ggggtcggcg gcgatgtcgg ctacccaccc 600 gacccgtctt gaaacacgga ccaaggagtc taacgcgtgc gcgagtcagg ggctcgcacg 660 aaagccgccg tggcgcaatg aaggtgaagg gccccgtccg ggggcccgag gtgggatcc 719 <210> 25 <211> 685 <212> DNA
<213> Mus musculus <400> 25 cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgtcgggg 60 aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ctgggcaggg 120 cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 180 cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 240 tttccctcag gatagctggc gctctcgcaa ccttcggaag cagttttatc cgggtaaagg 300 cggaatggat taggaggtct tggggccgga aacgatctca aactatttct caaactttaa 360 atgggtaagg aagcccggct cgctggcgtg gagccgggcg tggaatggaa gtgcctagtg 420 ggccactttt ggtaagcaga actggcgctg cgggatgaac cgaacgccgg gttaaggcgc 480 ccgatgccga cgctcatcag accccagaaa aggtgttggt tgatatagac agcaggacgg 540 tggccatgga agtcggaatc cgctaaggag tgtgtaacaa ctcacctgcc gaatcaacta 600 gccctgaaaa tggatggcgc tggagcgtcg ggcccatacc cggccgtcgc cggcagtcgg 660 aacgggacgg gacgggagcg gccgc 685 <210> 26 <211> 5162 <212> DNA
<213> Artificial Sequence <220>
<223> Chimeric bacterial plasmid <400> 26 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900 gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccac tagtaacggc 960 cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc 1020 ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaagaggat 1080 ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa 1140 aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact 1200 tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 1260 tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa 1320 s = +

cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc 1360 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1440 gagcattcct aggggtcttt cccctcctgc caaaggaatg caaggtctgt tgaatgtcgt 1500 gaaggaagca gttcctctg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1560 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1620 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1680 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1740 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1800 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1860 acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtg agcaagggcg 1920 aggagctgtt caccgtggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1980 acaagttcag cttgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2040 agttgatctg caccaccggc aagctgcccg tgccctggcc caccatcgtg accaccctga 2100 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2160 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gaggacggca 2220 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2280 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2340 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2400 tcaagatccg ccacaacatc gaggacggca gcgtcgagct cgccgaccac taccagcaga 2460 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2520 ccgCcCtgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2580 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagagctc 2640 gctgatcagc ctcgactgtg cctctagttg ctagccatct gttgtttgcc cctcccc.cgt 2700 gccttccttg accctggaag gtgccaatcc cactgtcctt tcctaataaa atgaggaaat 2760 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcatggcag 2820 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2880 ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac 2940 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 3000 catggtcata gctgtttcct gtgtgaaatt gttatcagct cacaattcca cacaacatac 3060 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 3120 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3180 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3240 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3300 cggtaatacg gttatccaca gaatcagggg ataaagcagg aaagaacatg tgagcaaaag 3360 gccagcaaaa ggccaggaac cgtaaaaagg ccgctttgct ggcgtttttc cataggctcc 3420 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3480 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3540 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3600 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3660 tgcacgaacc ccccgttcag ccctgccgct gcgccttatc cggtaactat cgtcttgagt 3720 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3780 gagcgaggca tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3840 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3900 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3960 agcagcagat tacccgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4020 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4080 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4140 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4200 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4260 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgatcac 4320 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4380 ctgcaacttt agccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4440 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4500 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4560 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4620 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4680 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4740 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4800 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4860 caaggatctt accgCtgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4920 cttcagcatc ttttactttc accagcgttt ctgggtgaac aaaaacagga aggcaaaatg 4980 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5040 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5100 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5160 tc 5162 <210> 27 <211> 5627 <212> DNA
<213> Artificial Sequence <220>
<223> pMG plasmid from InvivoGen; IRES sequence modified EMCV nucleotides 2736-3308 <400> 27 caccggcgaa ggaggcctag atctatcgat tgtacagcta gctcgacatg ataagataca 60 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 120 tttgtgatgc tattgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 180 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 240 gggggaggtg tgtgagttat tttaaaacaa gtaaaacctc tacaaatgtg gtagatccat 300 ttaaatgtta attaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 360 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 420 acgctcaagt caaaagtggc gaaacccgac aggactataa agataccagg cgtttccccc 480 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 540 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 600 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 660 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 720 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 780 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 840 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 900 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 960 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactct 1020 acgttaaggg attttggtca tggctagtta attaagctgc aataaacaat cattattttc 1080 attggatctg tgtgttggtt ttttgtgtgg gcttggggga gggggaggcc agaatgactc 1140 caagagctac aggaaggcag gtcagagacc ccactggaca aacagtggct ggactctgca 1200 ccataacaca caatcaacag gggagtgagc tggatcgagc tagagtccgt tacataactt 1260 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 1320 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 1380 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 1440 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 1500 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 1560 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 1620 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 1680 tgtcgtaaca actccgcccc attgacgcaa atgggcggta gccgtgtacg gtgggaggtc 1740 tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 1800 tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt 1860 ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc 1920 acccccttgg cttcttatgc atgctatact gtttttggct tggggtctat acacccccgc 1980 ttcctcatgt tataggtgat ggtatagctt agcctatagg tgtgggttat tgaccattat 2040 tgaccactcc cctattggtg acgatacttt ccattactaa tccataacat ggctctttgc 2100 cacaactctc tttattggct atatgccaat acactgtcct tcagagactg acacggactc 2160 tgtattttta caggatgggg tctcatttat tatttacaaa ttcacatata caacaccacc 2220 gtccccagtg cccgcagttt ttattaaaca taacgtggga tctccacgcg aatctcgggt 2280 acgtgttccg gacatgggct cttctccggt agcggcggag cttctacatc cgagccctgc 2340 tcccatgcct ccagcgactc atggtcgctc ggcagctcct tgctcctaac agtggaggcc 2400 agacttaggc acagcacgat gcccaccacc accagtgtgc cgcacaaggc cgtggcggta 2460 gggtatgtgt ctgaaaatga gctcggggag cgggcttgca ccgctgacgc atttggaaga 2520 cttaaggcag cggcagaaga agatgcaggc agctgagttg ttgtgttctg ataagagtca 2580 gaggtaactc ccgttgcggt gctgttaacg gtggagggca gtgtagtctg agcagtactc 2640 gttgctgccg cgcgcgccac cagacataat agctgacaga ctaacagact gttcctttcc 2700 atgggtcttt tctgcagtca cccgggggat ccttcgaacg tagctctaga ttgagtcgac 2760 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 2820 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 2880 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 2940 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 3000 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 3060 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 3120 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 3180 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 3240 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 3300 cgataatacc atgggtaagt gatatctact agttgtgacc ggcgcctagt gttgacaatt 3360 aatcatcggc atagtatatc ggcatagtat aatacgactc actataggag ggccatcatg 3420 tcgactacta accttcttct ctttcctaca gctgagatca ccggtaggag ggccatcatg 3480 . ' aaaaagcctg aattcaccgc gacgtctgtc gcgaagtttc tgatcgaaaa gttcgacagc 3540 gtctcccacc tgatgcagct ctcggagggc gaagaatctc gtgttttcag cttcgatgta 3600 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 3660 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg 3720 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 3780 gacctgcctg aaaccgaact gcccgctgtt ctgcaacccg tcgcggagct catggatgcg 3840 atcgctgcgg ccgatcttag ccagacgagc gggtttggcc cattcggacc gcaaggaatc 3900 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 3960 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 4020 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 4080 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 4140 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 4200 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcggcgcgg 4260 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 4320 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 4380 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 4440 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa 4500 tgagtcgaga attcgctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc 4560 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 4620 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 4680 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 4740 aagggggagg attgggaaga caatagcagg catgcgcagg gcccaattgc tcgagcggcc 4800 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgtaac 4860 taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc 4920 ccagtgcaag tgcaggtgcc agaacatttc tctatcgaag gatctgcgat cgctccggtg 4980 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 5040 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 5100 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 5160 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc 5220 gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc 5280 gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt 5340 taaagctcag gtcgagaccg ggcacttgtc cggcgctccc ttggagccta cctagactca 5400 gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt 5460 ctgttctgcg ccgttacaga tccaagctgt gaccggcgcc tacgtaagtg atatctacta 5520 gatttatcaa aaagagtgtt gacttgtgag cgctcacaat tgatacttag attcatcgag 5580 agggacacgt cgactactaa ccttcttctc tttcctacag ctgagat 5627 <210> 28 <211> 553 <212> DNA
<213> Artificial Sequence <220>
<223> pMG plasmid from InvivoGen: EMCV IRES sequence <400> 28 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt 60 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg 120 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc 180 gtgaaggaag cagttcctct ggaagcttct tgaa acaaa caacgtctgt agcgaccctt 240 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta 300 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg 360 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag 420 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac gtgtgtttag 480 tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa 540 cacgatgata ata 553 <210> 29 <211> 4692 <212> DNA
<213> Artificial Sequence <220>
<223> pDSredl-N1 plasmid from Clontech <400> 29 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 s cgttacataa cttacggtaa atggcccgcc tggctgaccg ccaaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtaccccc cctattgacg tgaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660 gatccaccgg tcgccaccat ggtgcgctcc tccaagaacg tcatcaagga gttcatgcgc 720 ttcaaggtgc gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc 780 gagggccgcc cctacgaggg ccacaacacc gtgaagctga aggagaccaa gggcggcccc 840 ctgcccttcg cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg 900 aagaagcccg ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg 960 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 1020 caggacggct gcttcatcga caaggtgaag ttcatcggcg tgaacttccc ctccgacggc 1080 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc 1140 gacggcgtgc tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac 1200 ctggtggagt tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac 1260 tacgtggact ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag 1320 tacgagcgca ccgagggccg ccaccacctg ttcctgtagc ggccacgagt ctagatcata 1380 atcacccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 1440 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 1500 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 1560 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt 1620 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1680 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1740 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1800 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 1860 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 1920 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 1980 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 2040 taatgggccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2100 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 2160 ataaaggctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg 2220 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2280 agcatgcatc tcaattagtc aacaaccagg tgtggaaagt ccccaggctc cccagcaggc 2340 agaagtatgc aaagtatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 2400 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 2460 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 2520 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc 2580 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 2640 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 2700 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 2760 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 2820 agctgtgctc gacttagtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 2880 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 2940 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3000 acatcgcatc gagcgatcac gtactc9gat ggaagccggt cttgtcgatc aggatgatct 3060 ggacga'agag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 3120 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3180 ggaaaatggc cgcttttctg gattcatcga ctttggccgg ctgggtgtgg cggaccgcta 3240 tcaggacata gcgttcgcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3300 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccctctatcg 3360 ccttcttgac gagttcttct gagcgggact ctggggttc aaatgaccga ccaagcgacg 3420 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3480 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 3540 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga 3600 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt 3660 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat 3720 tggggccaat acgcCCgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa 3780 ggcccagggc tcgcagccaa cgtcgagccg gcaggccctg ccatagcctc aggttactca 3840 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3900 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3960 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 4020 tgcttgccaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4080 i =, ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 4140 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 4200 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4260 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4320 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4380 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4440 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4500 agtcctgtcg ggtttcgcca cctctgacct gagcgtcgat ttttgtgatg ctcgtcaggg 4560 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4620 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4680 accgccatgc at 4692 <210> 30 <211> 4257 <212> DNA
<213> Artificial Sequence <220>
<223> pPur plasmid from Clontech <400> 30 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctgcccagc aggcagaagt 60 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 120 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 180 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 240 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 300 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttgcatg cctgcaggtc 360 ggccgccacg accggtgccg ccaccatccc ctgacccacg cccctgaccc ctcacaagga 420 gacgacgttc catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 480 cccgtgcctt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 540 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 600 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 660 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 720 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 780 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 840 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 900 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 960 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 1020 ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc 1080 atggctccga ccgaagccga cccgggcggc cccgccgacc ccgcacccgc ccccgaggcc 1140 caccgactct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 1200 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 1260 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1320 aataaagcat.ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1380 tatcatgtct ggatccccag gaagctcctc tgtgtcctca taaaccctaa cctcctctac 1440 ttgagaggac attccaatca taggctgccc atccaccctc tgtgtcctcc tgttaattag 1500 gtcacttaac aaaaaggaaa ttgggtaggg gtttttcaca gaccgctttc taagggtaat 1560 tttaaaatat ctgggaagtc ccttccactg ctgtgttcca gaagtgttgg taaacagccc 1620 acaaatgtca acagcagaaa catacaagct gtcagctttg cacaagggcc caacaccctg 1680 ctcatcaaga agcactgtgg ttgctgtgtt agtaatgtgc aaaacaggag gcacattttc 1740 cccacctgtg taggttccaa aatatctagt gttttcattt ttacttggat caggaaccca 1800 gcactccact ggataagcat tatccttatc caaaacagcc ttgtggtcag tgttcatctg 1860 ctgactgtca actgtagcat tttttggggt tacagtttga gcaggatatt tggtcctgta 1920 gtttgctaac acaccctgca gctccaaagg ttccccacca acagcaaaaa aatgaaaatt 1980 tgacccttga atgggttttc cagcaccatt ttcatgagtt ttttgtgtcc ctgaatgcaa 2040 gtttaacata gcagttaccc caataacctc agttttaaca gtaacagctt cccacatcaa 2100 aatatttcca caggttaagt cctcatttaa attaggcaaa ggaattcttg aagacgaaag 2160 ggcctdgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 2220 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 2280 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 2340 aaaaggaaga gtatgagtat tcaacatttc cgtttcgccc ttattccctt ttttgcggca 2400 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 2460 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 2520 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 2580 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcgt acactattct 2640 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2700 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2760 = =

ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 2820 gtaactcgcc ttgatcgttg ggaacaggag ctgaatgaag ccataccaaa cgacgagcgt 2880 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aaatattaac tggcgaacta 2940 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3000 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3060 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3120 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 3180 gagataggtg cctcactgat taagcattgg taactgtcag acgaaattta ctcatatata 3240 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3300 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3360 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3420 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3480 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 3540 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3600 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3660 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaccgggggg ttcgtgcaca 3720 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3780 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3840 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3900 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3960 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4020 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4080 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4140 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4200 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccag 4257 <210> 31 <211> 8136 <212> DNA
<213> Artificial Sequence <220>
<223> pWE15 cosmid vector <300>
<308> GenBank X65279 <309> 1995-04-14 <400> 31 ctatagtgag tcgtattatg cggccgcgaa ttcttgaaga cgaaagggcc tcgtgatacg 60 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 120 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 180 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 240 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gcttcctgtt 300 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 360 gtgggttaca tcgaactgga'tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 420 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 480 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 540 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 600 agtgctgcca taaccatgag tgataacact gctgccaact tacttctgac aacgatcgga 660 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 720 cgttgggaac cggagct as tgaagccata ccaaacgacg agcgtgacac cacgatgcct 780 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 840 cgcaaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 900 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 960 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 1020 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 1080 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 1140 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 1200 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 1260 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 1320 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 1380 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 1440 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 1500 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 1560 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 1620 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 1680 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 1740 cgagggagct ttcaggggga aacgcttggt atctttatag tcctgtcggg gtttcgccac 1800 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 1860 gccacgaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 1920 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 1980 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 2040 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 2100 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 2160 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 2220 cacgatcatg cgcacccgtc agatccagac atgataagat acattgatga gtttggacaa 2380 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2340 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2400 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2460 tgtggtatgg ctgattatga tctctagtca aggcactata catcaaatat tccttattaa 2520 cccctttaca aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag 2580 acactctatg cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc 2640 tacttataaa ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt 2700 cctttgtggt gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca 2760 aaaaacttag caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg 2820 gaggagtaga atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg 2880 agcaaaacag gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat 2940 aggttggaat ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta 3000 aaaattttat atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca 3060 caccacagaa gtaaggttcc ttcacaaaga tccggaccaa agcggccatc gtgcctcccc 3120 actcctgcag ttcgggggca tggatgcgcg gatagccgct gctggtttcc tggatgccga 3180 cggatttgca ctgccggtag aactcgcgag gtcgtccagc ctcaggcagc agctgaacca 3240 actcgcgagg ggatcgagcc cggggtgggc gaagaactcc agcatgagat ccccgcgctg 3300 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc 3360 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa 3420 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 3480 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 3540 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 3600 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 3660 tcgccatggg tcacgacgag atcctcgccg tcgggatgcg cgccttgagc ctggcgaaca 3720 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 3780 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 3840 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 3900 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 3960 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 4020 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct 4080 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 4140 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 4200 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 4260 atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 4320 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 4380 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 4440 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc 4500 accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg 4560 agtgcttgcg gcagcctaaa agctttttgc aaaagcctag gcctccaaaa aagcctcctc 4620 actacttctg gaatagctca gaggccgagg cggcctaaat aaaaaaaatt agtcagccat 4680 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg 4740 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc 4800 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc ataattctgc. 4860 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agccggatct 4920 gcaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat 4980 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc 5040 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 5100 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 5160 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cgcataaatc gccgtgacga 5220 tcagcggtcc aatgatcgaa gttaggctgg taagagcagc gagcgatcct tgaagctgtc 5280 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcggca tcccgatgcc 5340 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag 5400 caagacgtag cccagcgcgt cgggccgcca tgccggcgat aatggcctgc ttctcgccga 5460 aacgtttggt ggtgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 5520 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 5580 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 5640 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 5700 =

agggcatcgg tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 5760 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 5820 acagtccccc ggccacgggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 5880 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 5940 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct tggcagtcac 6000 agcatgcgca tatccatgct tcgaccatgc gctcacaaag taggtgaatg cgcaatgtag 6060 tacccacatc gtcatcgct"t tccactgctc tcgcgaataa agatggaaaa tcaatctcat 6120 ggtaatagtc catgaaaatc cttgtattca taaatcctcc aggtagctat atgcaaattg 6180 aaacaaaaga gatggtgatc tttctaagag atgatggaat ctcccttcag tatcccgatg 6240 gtcaatgcgc tggatatggg atagatggga atatgctgat ttttatggga cagagttgcg 6300 aacttttgcc aactaaaatc attttgcacg atcagcgcac tacgaacttt acccacaaat 6360 agtcaggtaa tgaatcctga tataaagaca ggttgataaa tcagtcttct acgcgcatcg 6420 cacgcgcaca ccgtagaaag tctttcagtt gtgagcctgg gcaaaccgtt aactttcggc 6480 ggctttgctg tgcgacaggc tcacgtttaa aaggaaataa atcatgggtc ataaaattat 6540 cacgttgtcc ggcgcggcga cggatgttct gtatgcgctg tttttccgtg gcgcgttgct 6600 gtctggtgat ctgccttcta aatctggcac agccgaattg cgcgagcttg gttttgctga 6660 aaccagacac acagcaactg aataccagaa agaaaatcac tttacctttc tgacatcaga 6720 agggcagaaa tttgccgttg aacacctggt caatacgcgt tttggtgagc agcaatattg 6780 cgcttcgatg acgcttggcg ttgagattga tacctctgct gcacaaaagg caatcgacga 6840 gctggaccag cgcattcgtg acaccgtctc cttcgaactt attcgcaatg gagtgtcatt 6900 catcaaggac gccgctatcg caaatggtgc tatccacgca gcggcaatcg aaacacctca 6960 gccggtgacc aatatctaca acatcagcct tggtatccag cgtgatgagc cagcgcagaa 7020 caaggtaacc gtcagtgccg ataagttcaa agttaaacct ggtgttgata ccaacattga 7080 aacgttgatc gaaaacgcgc tgaaaaacgc tgctgaatgt gcggcgctgg atgtcacaaa 7140 gcaaatggca gcagacaaga aagcgatgga tgaactggct tcctatgtcc gcacggccat 7200 catgatggaa tgtttccccg gtggtgttat ctggcagcag tgccgtcgat agtatgcaat 7260 tgataattat tatcatttgc gggtcctttc cggcgatccg ccttgttacg gggcggcgac 7320 ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg ttcttcttcg 7380 tcataactta atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg acaggtgctg 7440 aaagcgagct ttttggcctc tgtcgtttcc tttctctgtt tttgtccgtg gaatgaacaa 7500 tggaagtcaa caaaaagcag ctggctgaca ttttcggtgc gagtatccgt accattcaga 7560 actggcagga acagggaatg cccgttctgc gaggcggtgg caagggtaat gaggtgcttt 7620 atgactctgc cgccgtcata aaatggtatg ccgaaaggga tgctgaaatt gagaacgaaa 7680 agctgcgccg ggaggttgaa gaactgcggc aggccagcga ggcagatcca caggacgggt 7740 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg 7800 cggcggcaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg 7860 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc 7920 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg 7980 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc 8040 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagcggt caaacatgag 8100 aattcgcggc cgcaattaac cctcactaaa ggatcc 8136 <210> 32 <211> 2713 <212> DNA
<213> Artificial Sequence <220>
<223> pNEB193 plasmid <400> 32 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaattaag cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgggggc 420 gcgccggatc cttaattaag tctagagtcg actgtttaaa cctgcaggca tgcaagcttg 480 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 540 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 600 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 660 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 720 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 780 tcaaaggcgg taatacggtt atccacaaaa tcaggggata acgcaggaaa gaacatgtga 840 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 900 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 960 = =

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1020 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1080 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1140 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 1200 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 1260 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 1320 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 1380 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 1440 gtttgcaagc agcagattac gcgcaaaaaa aaaggatctc aagaagatcc tttgatcttt 1500 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1560 ttatcaaaaa ggatcttaac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 1620 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 1680 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 1740 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 1800 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 1860 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 1920 gtaagtagtt cgccagttaa tagtttgcgc aacgtttctg ccattgctac aggcatcgtg 1980 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg'gttcccaacg atcaaggcga 2040 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 2100 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 2160 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 2220 ttctgagaat agtgtatgcg gcgaccgagt tgctctcggc cggcgtcaat acgggataat 2280 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 2340 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 2400 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 2460 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 2520 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 2580 gaatgtattt agaaaaataa acaaataggg gttccgccca catttccccg aaaagtgcca 2640 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 2700 aggccctttc gtc 2713 <210> 33 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> attP
<400> 33 cagctttttt atactaagtt g 21 <210> 34 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> attB
<400> 34 ctgctttttt atactaactt g 21 <210> 35 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> attL
<400> 35 ctgctttttt atactaagtt g 21 <210> 36 <211> 21 <212> DNA

<213> Artificial Sequence <220>
<223> attR
<400> 36 cagctttttt atactaactt g 21 <210> 37 <211> 1071 <212> DNA
<213> Artificial Sequence <220>
<223> Integrase E174R
<221> CDS
<222> (1)...(1071) <223> Nucleotide sequence encoding Integrase E147R
<400> 37 atg gga aga agg cga agt cat gag cgc cgg gat tta ccc cct aac ctt 48 Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu tat ata aga aac aat gga tat tac tgc tac agg gac cca agg acg ggt 96 Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly aaa gag ttt gga tta ggc aga gac agg cga atc gca atc act gaa get 144 Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala ata cag gcc aac att gag tta ttt tca gga cac aaa cac aag cct ctg 192 Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu aca gcg aga atc aac agt gat aat tcc gtt acg tta cat tca tgg ctt 240 Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu gat cgc tac gaa aaa atc ctg gcc agc aga gga atc aag cag aag aca 288 Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr ctc ata aat tac atg agc aaa att aaa gca ata agg agg ggt ctg cct 336 Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro gat get cca ctt gaa gac atc acc aca aaa gaa att gcg gca atg ctc 384 Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu aat gga tac ata gac gag ggc aag gcg gcg tca gcc aag tta atc aga 432 Asn Gly Tyr Ile Asp Glu Gly Lys Ala Ala Ser Ala Lys Leu Ile Arg tca aca ctg agc gat gca ttc cga gag gca ata get gaa ggc cat ata 480 Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile aca aca aac cat gtc get gcc act cgc gca gca aaa tct aga gta agg 528 Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg aga tca aga ctt acg get gac gaa tac ctg aaa att tat caa gca gca 576 Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala gaa tca tca cca tgt tgg ctc aga ctt gca atg gaa ctg get gtt gtt 624 Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val acc ggg caa cga gtt ggt gat tta tgc gaa atg aag tgg tct gat atc 672 Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile gta gat gga tat ctt tat gtc gag caa agc aaa aca ggc gta aaa att 720 Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile gcc atc cca aca gca ttg cat att gat get ctc gga ata tca atg aag 768 Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys gaa aca ctt gat aaa tgc aaa gag att ctt ggc gga gaa acc ata att 816 Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile gca tct act cgt cgc gaa ccg ctt tca tcc ggc aca gta tca agg tat 864 Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr ttt atg cgc gca cga aaa gca tca ggt ctt tcc ttc gaa ggg gat ccg 912 Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro cct acc ttt cac gag ttg cgc agt ttg tct gca aga ctc tat gag aag 960 Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys cag ata agc gat aag ttt get caa cat ctt ctc ggg cat aag tcg gac 1008 Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp acc atg gca tca cag tat cgt gat gac aga ggc agg gag tgg gac aaa 1056 Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys att gaa atc aaa taa 1071 Ile Glu Ile Lys <210> 38 <211> 356 <212> PRT
<213> Artificial Sequence <220>
<223> Integrase E147R
<400> 38 Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu Asn Gly Tyr Ile Asp Glu G1y Lys Ala Ala Ser Ala Lys Leu Ile Arg Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys Ile Glu Ile Lys <210> 39 <211> 876 <212> DNA
<213> Discosoma species <220>
<221> CDS
<222> (45) ... (737) <223> Nucleotide sequence encoding red flourescent protein (FP593) <300>
<308> GenBank AF272711 <309> 2000-09-26 <400> 39 agtttcagcc agtgacaggg tgagctgcca ggtattctaa caag atg agt tgt tcc 56 Met Ser Cys Ser I
aag aat gtg atc aag gag ttc atg agg ttc aag gtt cgt atg gaa gga 104 Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly acg gtc aat ggg cac gag ttt gaa ata aaa ggc gaa ggt gaa ggg agg 152 Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu Gly Glu Gly Arg cct tac gaa ggt cac tgt tcc gta aag ctt atg gta acc aag ggt gga 200 Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val Thr Lys Gly Gly cct ttg cca ttt get ttt gat att ttg tca cca caa ttt cag tat gga 248 Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly agc aag gta tat gtc aaa cac cct gcc gac ata cca gac tat aaa aag 296 Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys ctg tca ttt cct gag gga ttt aaa tgg gaa agg gtc atg aac ttt gaa 344 Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu gac ggt ggc gtg gtt act gta tcc caa gat tcc agt ttg aaa gac ggc 392 Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser Leu Lys Asp Gly tgt ttc atc tac gag gtc aag ttc att ggg gtg aac ttt cct tct gat 440 Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp gga cct gtt atg cag agg agg aca cgg ggc tgg gaa gcc agc tct gag 488 Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu Ala Ser Ser Glu cgt ttg tat cct cgt gat ggg gtg ctg aaa gga gac atc cat atg get 536 Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp Ile His Met Ala ctg agg ctg gaa gga ggc ggc cat tac ctc gtt gaa ttc aaa agt att 584 Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu Phe Lys Ser Ile tac atg gta aag aag cct tca gtg cag ttg cca ggc tac tat tat gtt 632 Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly Tyr Tyr Tyr Val gac tcc aaa ctg gat atg acg agc cac aac gaa gat tac aca gtc gtt 680 Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp Tyr Thr Val Val gag cag tat gaa aaa acc cag gga cgc cac cat ccg ttc att aag cct 728 Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro Phe Ile Lys Pro ctg cag tga actcggctca gtcatggatt agcggtaatg gccacaaaag 777 Leu Gln gcacgatgat cgttttttag gaatgcagcc aaaaattgaa ggttatgaca gtagaaatac 837 aagcaacagg ctttgcttat taaacatgta attgaaaac 876 <210> 40 <211> 230 <212> PRT
<213> Discosoma species <400> 40 Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln Phe Gin Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Ser Gin Asp Ser Ser Leu Lys Asp Gly Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu Ala Ser Ser Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp Ile His Met Ala Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu Phe Lys Ser Ile Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp Tyr Thr Val Val Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro Phe Ile Lys Pro Leu Gln <210> 41 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> m-att;

<221> misc difference <222> 18 <223> n is a or g or c or t/u <400> 41 rkycwgcttt yktrtacnaa stsgb 25 <210> 42 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> m-attB;

<221> misc difference <222> 18 <223> n is a or g or c or t/u <400> 42 agccwgcttt yktrtacnaa ctsgb 25 <210> 43 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> m-attR

<221> misc_difference <222> 18 <223> n is a or g or c or t/u =

<400> 43 gttcagcttt cktrtacnaa ctsgb 25 <210> 44 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> m-attL

<221> misc_difference <222> 18 <223> n is a or g or c or t/u <400> 44 agccwgcttt cktrtacnaa gtsgb 25 <210> 45 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> m-attPl <221> misc_difference <222> 18 <223> n is a or g or c or t/u <400> 45 gttcagcttt yktrtacnaa gtsgb 25 <210> 46 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attBl <400> 46 agcctgcttt tttgtacaaa cttgt 25 <210> 47 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attB2 <400> 47 agcctgcttt cttgtacaaa cttgt 25 <210> 48 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attB3 <400> 48 acccagcttt cttgtacaaa cttgt 25 <210> 49 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attRl <400> 49 gttcagcttt tttgtacaaa cttgt 25 <210> 50 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attR2 <400> 50 gttcagcttt cttgtacaaa cttgt 25 <210> 51 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attR3 <400> 51 gttcagcttt cttgtacaaa gttgg 25 <210> 52 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attL1 <400> 52 agcctgcttt tttgtacaaa gttgg 25 <210> 53 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attL2 <400> 53 agcctgcttt cttgtacaaa gttgg 25 <210> 54 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attL3 <400> 54 acccagcttt cttgtacaaa gttgg 25 <210> 55 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attPl <400> 55 gttcagcttt tttgtacaaa gttgg 25 <210> 56 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> attP2,P3 <400> 56 gttcagcttt cttgtacaaa gttgg 25 <210> 57 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<223> Lox P site <400> 57 ataacttcgt ataatgtatg ctatacgaag ttat 34 <210> 58 <211> 1032 <212> DNA
<213> Escherichia coli <220>
<221> CDS
<222> (1)...(1032) <223> nucleotide sequence encoding Cre recombinase <400> 58 atg tcc aat tta ctg acc gta cac caa aat ttg cct gca tta ccg gtc 48 Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val gat gca acg agt gat gag gtt cgc aag aac ctg atg gac atg ttc agg 96 Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg gat cgc cag gcg ttt tct gag cat acc tgg aaa atg ctt ctg tcc gtt 144 Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val tgc cgg tcg tgg gcg gca tgg tgc aag ttg aat aac cgg aaa tgg ttt 192 Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe ccc gca gaa cct gaa gat gtt cgc gat tat ctt cta tat ctt cag gcg 240 Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala cgc ggt ctg gca gta aaa act atc cag caa cat ttg ggc cag cta aac 288 Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn atg ctt cat cgt cgg tcc ggg ctg cca cga cca agt gac agc aat get 336 =

Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala gtt tca ctg gtt atg cgg cgg atc cga aaa gaa aac gtt gat gcc ggt 384 Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly gaa cgt gca aaa cag get cta gcg ttc gaa cgc act gat ttc gac cag 432 Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln gtt cgt tca ctc atg gaa aat agc gat cgc tgc cag gat ata cgt aat 4B0 Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn ctg gca ttt ctg ggg att get tat aac acc ctg tta cgt ata gcc gaa 528 Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu att gcc agg atc agg gtt aaa gat atc tca cgt act gac ggt ggg aga 576 Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg atg tta atc cat att ggc aga acg aaa acg ctg gtt agc acc gca ggt 624 Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly gta gag aag gca ctt agc ctg ggg gta act aaa ctg gtc gag cga tgg 672 Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp att tcc gtc tct ggt gta get gat gat ccg aat aac tac ctg ttt tgc 720 Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cvs cgg gtc aga aaa aat ggt gtt gcc gcg cca tct gcc acc agc cag cta 768 Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu tca act cgc gcc ctg gaa ggg att ttt gaa gca act cat cga ttg att 816 Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile tac ggc get aag gat gac tct ggt cag aga tac ctg gcc tgg tct gga 864 Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly cac agt gcc cgt gtc gga gcc gcg cga gat atg gcc cgc get gga gtt 912 His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val tca ata ccg gag atc atg caa get ggt ggc tgg acc aat gta aat att 960 Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile gtc atg aac tat atc cgt aac ctg gat agt gaa aca ggg gca atg gtg 1008 Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val cgc ctg ctg gaa gat ggc gat tag 1032 Arg Leu Leu Glu Asp Gly Asp <210> 59 <211> 343 <212> PRT

= =

<213> Escherichia coli <400> 59 Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile Tyr Gly Ala Lys Asp Asp Ser Gly Gin Arg Tyr Leu Ala Trp Ser Gly His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val Arg Leu Leu Glu Asp Gly Asp <210> 60 <211> 1272 <212> DNA
<213> Saccharomyces cerevisiae <220>
<221> CDS
<222> (1)...(1272) <223> nucleotide sequence encoding Flip recombinase <400> 60 atg cca caa ttt ggt ata tta tgt aaa aca cca cct aag gtg ctt gtt 48 Met Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val cgt cag ttt gtg gaa agg ttt gaa aga cct tca ggt gag aaa ata gca 96 Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala tta tgt get gat gaa cta acc tat tta tgt tgg atg att aca cat aac 144 Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn gga aca gca atc aag aga gcc aca ttc atg agc tat aat act atc ata 192 Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile agc aat tcg ctg agt ttc gat att gtc aat aaa tca ctc cag ttt aaa 240 Ser Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys tac aag acg caa aaa gca aca att ctg gaa gcc tca tta aag aaa ttg 288 Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu att act get tgg gaa ttt aca att att cat tac tat gga caa aaa cat 336 Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gin Lys His caa tat gat atc act gat att gta agt agt ttg caa tta cag ttc gaa 384 Gln Ser Asp Ile Thr Asp Ile Val Her Her Leu Gln Leu Gln Phe Glu tca tag gaa gaa gca gat aag gga aat agc cac agt aaa aaa atg ctt 432 Ser Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu aaa pea ctt cta agt gag ggt gaa agc atc tgg gag atc act gag aaa 480 Lys Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys ata cta aat tag ttt gag tat act tcg aga ttt aca aaa aca aaa act 528 Ile Leu Asn Her Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr tta tac caa ttc ctc ttc cta gat act ttc atc aat tgt gga aga ttc 576 Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe agc gat att aag aac gtt gat ccg aaa tca ttt aaa tta gtc caa aat 624 Ser Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn aag tat ctg gga gta ata atc cag tgt tta gtg aca gag aca aag aca 672 Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr agc gtt agt agg cac ata tac ttc ttt agc gca agg ggt agg atc gat 720 Ser Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp cca ctt gta tat ttg gat gaa ttt ttg agg aat tat gaa cca gtc cta 768 Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu aaa cga gta aat agg acc ggc aat tat tca agc aat aaa cag gaa tac 816 Lys Arg Val Asn Arg Thr Gly Asn Her Ser Ser Asn Lys Gln Glu Tyr caa tta tta aaa gat aac tta gtc aga tag tac aat aaa gat ttg aag 864 Gln Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys aaa aat gcg act tat tca atc ttt get ata aaa aat ggc cca aaa tat 912 Lys Asn Ala Pro Tyr Her Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser = = ' cac att gga aga cat ttg atg acc tca ttt ctt tca atg aag ggc cta 960 His Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu acg gag ttg act aat gtt gtg gga aat tgg age gat aag cgt get tct 1008 Thr Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser gcc gtg gcc agg aca acg tat act cat cag ata aca gca ata cct gat 1056 Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp cac tac ttc gca cta gtt tct cgg tac tat gca tat gat cca ata tca 1104 His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser aag gaa atg ata gca ttg aag gat gag act aat cca att gag gag tgg 1152 Lys Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp cag cat ata gaa cag cta aag ggt agt get gaa gga agc ata cga tac 1200 Gln His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr ccc gca tgg aat gag ata ata tca cag gag gta cta gac tac ctt tca 1248 Pro Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser tcc tac ata aat aga cgc ata taa 1272 Ser Tyr Ile Asn Arg Arg Ile <210> 61 <211> 422 <212> PRT
<213> Saccharomyces cerevisiae <400> 61 Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile Ser Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His Gln Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu Ser Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu Lys Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys Ile Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe Ser Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr Ser ' =

Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu Lys Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gin Glu Tyr Gin Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys Lys Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser His Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu Thr Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser Lys Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp Gln His Ile Glu Gin Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr Pro Ala Trp Asn Gly Ile Ile Ser Gin Glu Val Leu Asp Tyr Leu Ser Ser Tyr Ile Asn Arg Arg Ile <210> 62 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> IR2 <400> 62 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 48 <210> 63 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> IR1 <400> 63 gaagttccta tactttctag agaataggaa cttcggaata ggaacttc 48 <210> 64 <211> 66 <212> DNA
<213> Bacteriophage mu <220>
<221> CDS
<222> (1) ... (66) <223> nucleotide sequence encoding GIN recombinase <400> 64 tca act ctg tat aaa aaa cac ccc gcg aaa cga gcg cat ata gaa aac 48 Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn gac gat cga atc aat taa 66 Asp Asp Arg Ile Asn *

=

<210> 65 <211> 21 <212> PRT
<213> bacteriophage mu <400> 65 Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn Asp Asp Arg Ile Asn <210> 66 <211> 69 <212> DNA
<213> Bacteriophage mu <220>
<221> CDS
<222> (1) ... (69) <223> nucleotide sequence encoding Gin recombinase <400> 66 tat aaa aaa cat ccc gcg aaa cga acg cat ata gaa aac gac gat cga 48 Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg atc aat caa atc gat cgg taa 69 Ile Asn Gln Ile Asp Arg <210> 67 <211> 22 <212> PRT
<213> bacteriophage mu <220>
<223> Gin recombinase of bacteriophage mu <400> 67 Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg Ile Asn Gin Ile Asp Arg <210> 68 <211> 555 <212> DNA
<213> Escherichia coli <220>
<221> CDS
<222> (1)...(555) <223> nucleotide sequence encoding PIN recombinase <400> 68 atg ctt att ggc tat gta cgc gta tca aca aat gac cag aac aca gat 48 Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asn Asp Gln Asn Thr Asp cta caa cgt aat gcg ctg aac tgt gca gga tgc gag ctg att ttt gaa 96 Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu =

gac aag ata agc ggc aca aag tcc gaa agg ccg gga ctg aaa aaa ctg 144 Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu ctc agg aca tta tcg gca ggt gac act ctg gtt gtc tgg aag ctg gat 192 Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp cgg ctg ggg cgt agt atg cgg cat ctt gtc gtg ctg gtg gag gag ttg 240 Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu cgc gaa cga ggc atc aac ttt cgt agt ctg acg gat tca att. gat acc 288 Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr agc aca cca atg gga cgc ttt ttc ttt cat gtg atg ggt gcc ctg get 336 Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala gaa atg gag cgt gaa ctg att gtt gaa cga aca aaa get gga ctg gaa 384 Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu act get cgt gca cag gga cga att ggt gga cgt cgt ccc aaa ctt aca 432 Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr cca gaa caa tgg gca caa get gga cga tta att gca gca gga act cct 480 Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro cgc cag aag gtg gcg att atc tat gat gtt ggt gtg tca act ttg tat 528 Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr aag agg ttt cct gca ggg gat aaa taa 555 Lys Arg Phe Pro Ala Gly Asp Lys <210> 69 <211> 184 <212> PRT
<213> Escherichia coli <400> 69 Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asia Asp Gln Asn Thr Asp Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro = w.

Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr Lys Arg Phe Pro Ala Gly Asp Lys <210> 70 <211> 4778 <212> DNA
<213> Artificial Sequence <220>
<223> pcx plasmid <400> 70 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccaac cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccggggcc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgaaccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgctccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctgacgt gtgaccggcg 1620 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcactcct caggtgcagg 1740 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactaag 1800 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt 1860 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 1920 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt 1980 agagtttggc aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt 2040 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact 2100 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa 2160 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata 2220 gctgtccctc ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg 2280 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 2340 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 2400 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc 2460 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 2520 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 2580 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 2640 ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2700 tcacaaattt cacaaataaa gcattttttt cattgcattc tagttgtggt ttgtccaaac 2760 tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg 2820 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2880 ggtcgttcgg ctgcggcgag cgttatcagc tcactcaaag gcggtaatac ggttatccac 2940 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 3000 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 3060 caaaaatcga cgctcaagtc aaaggtggcg aaacccgaca ggactataaa gataccaggc 3120 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttacggtata 3180 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 3240 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 3300 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 3360 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3420 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 3480 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3540 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3600 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3660 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3720 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3780 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 3840 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3900 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3960 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 4020 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 4080 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 4140 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 4200 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 4260 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 4320 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 4380 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 4440 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4500 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4560 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4620 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4680 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4740 aggggttccg cgcacatttc cccgaaaagt gccacctg 4778 <210> 71 <211> 5510 <212> DNA
<213> Artificial Sequence <220>
<223> pCXeGFP plasmid <400> 71 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800 . =

acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggctcacc ctcgtgacca 1920 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg cacCatcttc ttcaaggacg 2040 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2340 cccaatccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520 aataccactg aaatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640 ttttgtgtct ctcactcgga aggacatatg ggaaggcaaa tcatttaaaa catcagaatg 2700 agtatttggt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880 acatccctaa aattttcctt acatttttta ctagccagat ttttcctcct ctcctgacta 2940 ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360 ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat 3420 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3480 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg 3540 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3600 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3660 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3720 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3780 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3840 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3900 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 3960 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4020 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4080 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4140 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4200 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4260 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4320 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560 tctatttggt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataatacgg cgccacatag 5160 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220.
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280 attttttatt ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5510 <210> 72 <211> 282 <212> DNA
<213> Artificial Sequence = =

<220>
<223> attp <400> 72 ccttgcgcta atgctctgtt acaggtcact aataccatct aagtagttga ttcatagtga 60 ctgcatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa 120 tatattgata tttatatcat tttacgtttc tcgttcagct tttttatact aagttggcat 180 tataaaaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa 240 aatcattatt tgatttcaat tttgtcccac tccctgcctc tg 282 <210> 73 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 73 ggccccgtaa tgcagaagaa 20 <210> 74 <211>-32 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 74 ggtttaaagt gcgctcctcc aagaacgtca tc 32 <210> 75 <211> 40 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 75 agatctagag ccgccgctac aggaacaggt ggtggcggcc 40 <210> 76 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Primer SPacSV40 <400> 76 ctgttaatta actgtggaat gtgtgtcagt tagggtg 37 <210> 77 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer Antisense Zeo <400> 77 tgaacagggt cacgtcgtcc 20 <210> 78 <211> 24 = =

<212> DNA
<223> Artificial Sequence <220>
<223> Primer 5' NETS
<400> 78 gggccgaaac gatctcaacc tatt 24 <210> 79 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Primer 3' HETS
<400> 79 cgcagcggcc ctcctactc 19 <210> 80 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Primer SBSD
<400> 80 accatgaaaa catttaacat ttctcaaca 29 <210> 81 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Primer SV40polyA
<400> 81 tttatttgtg aaatttgtga tgctattgc 29 <210> 82 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer 3BSP
<400> 82 ttaatttcgg gtatatttga gtgga 25 <210> 83 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Primer EPOSXBA
<400> 83 tatctagaat gggggtgcac gaatgtcctg cc 32 <210> 84 <211> 32 <212> DNA

<213> Artificial Sequence <220>
<223> Primer EP03SBI
<400> 84 tacgtacgtc atctgtcccc tgtcctgcag gc 32 <210> 85 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Primer GENEP03BSI
<400> 85 cgtacgtcat ctgtcccctg tcctgca 27 <210> 86 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Primer GENEPOSXBA
<400> 86 tctagaatgg gggtgcaggg tgagtact 28 <210> 87 <211> 4862 <212> DNA
<213> Artificial Sequence <220>
<223> pD2eGFP-1N plasmid from Clontech <400> 87 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacgtcaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 720 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 780 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 840 cccgtgccct ggcccaccct cctgaccacc ctgacctacg gcgtgcagtg cttcagccgc 900 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 960 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1020 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1080 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1140 gccgacaagc agaagaacgg catcaaggtg aactacaaga tccgccacaa catcgaggac 1200 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1260 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1320 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 1380 gacgagctgt acaagaagct tagccatggc ttcccgccgg aggtggagga gcaggatgat 1440 ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg accgtcaccc tgcagcctgt 1500 gctactgcta ggatcaatgt gtagatgcgc ggccgcgact ctagatcata atcagecata 1560 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 1620 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 1680 = =

aataaagcaa tagCatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 1740 gtcgtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg 1800 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 1860 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 1920 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 1980 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 2040 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 2100 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 2160 ctggcaagtg tagcgatcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 2220 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2280 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 2340 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc 2400 agttagggtg tggaaagtcc ccaggCtccc cagcaggcag aagtatgcaa agcatgcatc 2460 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2580 ccctaactcc gcccatttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2640 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 2700 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg 2760 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 2820 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 2880 caaggccgcc cagttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 2940 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3000 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3060 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3120 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3180 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3240 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 3300 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 3360 cgcttttctg gattcatcga ctgtggccgg ctgggggtgg cggaccgcta tcaagagata 3420 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 3480 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 3540 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 3600 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 3660 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 3720 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta 3780 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg 3840 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 3900 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc 3960 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt 4020 agattgattt aaaacttcat ttttaattta aaaggatCta ggtgaagatc ctttttgata 4080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 4140 aaaagatcaa aggatcttct taatatcctt tttttctgcg cgtaatctgc tgcttgcaaa 4200 caaaaaaacc accgctacca gcggtggtct gtttgccgga tcaagagcta ccaactcttt 4260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 4320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 4380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 4440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 4500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 4560 gcgccacgct ttccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 4620 caggagagCg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 4680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 4740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 4800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc 4860 at 4862 <210> 88 <211> 5192 <212> DNA
<213> Artificial Sequence <220>
<223> pIRESpuro2 plasmid from Clontech <400> 88 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tgtctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag ggttatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt 960 ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta 1020 attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga 1080 cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg 1140 cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt 1200 caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca 1260 ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc 1320 tagggcgggc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc 1380 ttggaataag gccggtgtgc gtttgtctat atgtgatttt ccaccatatt gccgtctttt 1440 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt 1500 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 1560 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 1620 cctggcgaca ggtgcctctg cgaccaaaag ccacgtgtat aagatacacc tgcaaaggcg 1680 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 1740 tcaagcgtat tcaacaaggg gctgaagaat gcccacaagg taccccattg tatgggatct 1800 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 1860 ggccccctga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac 1920 aacccacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc 1980 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 2040 cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct 2100 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 2160 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 2220 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 2280 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 2340 actaccaggg caaaggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 2400 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 2460 ggctcggctt caccgtcacc gccgacgtcg agtgcccgaa ggaccgcgcg acctggtgca 2520 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga 2580 gcgcacgacc ccatggctcc gaccgaagcc gacccgggcg gccccgccga ccccccaccc 2640 gcccccgagg cccaccaact ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 2700 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 2760 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 2820 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2880 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2940 gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 3000 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 3060 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3120 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3180 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3240 tgcgtattgg gcgctcttcc gcttcctctc tcactgactc gctgcgctcg gtcgttcggc 3300 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3360 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3420 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3480 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3540 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3600 ttctcccttc gggaagcgtg gctctttctc aatgctcacg ctgtaggtat ctcagttcgg 3660 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3720 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacaCgac ttatcgccac 3780 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3840 tettgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3900 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3960 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4020 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4080 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4140 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4200 S =' aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4260 ccagactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4320 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4380 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4440 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4500 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4560 ccggttCcca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4620 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4680 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4740 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4800 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4860 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4920 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4980 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5040 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5100 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5160 gcacatttcc ccgaaaagtg ccacctgacg tc 5192 <210> 89 <211> 11182 <212> DNA
<213> Artificial Sequence <220>
<223> pAgl Plasmid <400> 89 catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60 atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120 agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180 gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240 agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300 ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360 ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420 acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480 ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540 acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600 agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660 tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720 tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780 ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840 gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900 gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960 cgaagagatc gaggcagaaa tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020 ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080 gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140 tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200 aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260 aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320 ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380 ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440 cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500 atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560 accgcggaac tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620 gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 16B0 gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740 ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800 cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860 aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920 gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980 agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa gacaagatca 2040 ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100 atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160 accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220 tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280 cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340 gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400 tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460 cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520 gatgctctat gacgtgggca cccgcgaaag tcggagcatc atggacgtgg ccgttttccg 2580 tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640 cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700 gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760 gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820 tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttataca ccacgcacgt 2880 tgccatgcag cctacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940 agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000 gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060 gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120 ggcacgccgc gccgcaggga aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180 cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240 aaatgacctg ccggagtacg atttgaagga ggaggcgggg caagctgccc cgatcctagt 3300 catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360 gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420 tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480 cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540 aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600 ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660 gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720 aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780 actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840 aaaacctctg acccatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggttgttcg 4140 gatgtggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aagcccagga accgtaaaaa 4260 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320 acgctcaagt cagagggggc gaaacccgac aggactataa agataccagg cgtttccccc 4380 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920 acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980 atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040 ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100 gtccgccctg ccgcttcttc caagatcaat aaagccactt actttgccat ctttcacaaa 5160 gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220 ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280 gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340 taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400 cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460 gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520 gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580 atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640 tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700 tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760 tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820 aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880 ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940 caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000 gtttcaaacc cggcagctta gttgccgttc ttccgaatag catccgttac atgagcaaag 6060 tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120 cgagtggtga ttttgtgccg agctgccggt cgaggagctg ttggctggct ggtggcagga 6180 tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240 taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300 gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360 ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420 s-46-ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480 ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540 ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600 atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccactctctt gaagccctgt 6660 gcctccaggg atttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720 cggggggaga cgtacacggt cgactcggcc gtccagcttt aggcgttgcg tgccttccag 6780 gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840 cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900 aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960 gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020 gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa taaaatgaac 7080 ttccttatat aaaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140 agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200 cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260 aacgaaggcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320 tgtccttttg atgaagtgac agatagctgg gcaatggaat'ccgaggaggt ttcccgatat 7380 taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440 cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500 agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560 gggaccactg tcggcagagg catcttgaac gatagccttt cctttatgtc aatgatggca 7620 tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680 atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740 gtcttctgag actgtatgtt tgatattctt gcagtagacg agagtgtcgt gctccaccat 7800 gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040 tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280 tccgaagccc aacctttcat agaaggcggc gttggaatcg aaatctcgta gcacgtgtca 8340 gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 8400 ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460 cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 8520 ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580 gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640 gttccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700 caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760 gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820 accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880 attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940 aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000 ccaaagatgg acccccaccc acgaggagca tcgtagaaaa agaagacgtt ccaaccacgt 9060 cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtttactcca 9120 aaagtctcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180 taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240 cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggccatcg 9300 ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360 tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420 ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480 aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta=caaatctatc 9540 tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggaggtcgt ggatatgtcc 9720 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcattttg 9780 catcggccgc gctcccgatt CCggaagtgc ttgacattgg ggagtttagc gagagcctga 9840 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agaccttcct gaaaccgaac 9900 tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 10260 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320 acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560 gtggaaaccg acgCCccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620 atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680 ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740 gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800 agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttggcact 10860 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 10920 tgcagcacat ccccctttcg caagctgccg taatagcgaa gaggcccgca ccgatcgccc 10980 ttcccaacag ttgcgcagcc tgaatggcga atgctagagc agcttgagct tggatcagat 11040 tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 11100 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 11160 tccgttcgtc catttgtatg tg 11182 <210> 90 <211> 8428 <212> DNA
<213> Artificial Sequence <220>
<223> pCambia3300 Plasmid <400> 90 catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60 atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120 agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180 gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240 agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300 ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360 ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420 acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480 ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540 acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600 agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660 tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720 tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780 ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840 gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaattaac 900 gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960 cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020 ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080 gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140 tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200 aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260 aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320 ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380 ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440 cggcgccact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500 atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560 accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620 gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680 gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740 ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800 cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860 aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920 gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980 agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040 ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100 atgagtagat gaattttagc gcctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160 accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220 tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280 cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340 gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400 tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tccctgcaac cgccggcagc 2460 cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520 gatgctctat gagctggcca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580 tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640 cgtagaggtt tccgcagggc cggccggcat ggccagtggg tgggattacg acctggtact 2700 gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760 gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcaagccga 2820 tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880 tcacatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940 agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000 gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060 gacggttcac cccgattact ttttgatcga tcccggcatc gccagttttc tctaccgcct 3120 ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180 cagtggcagc gccggagagt tcaagaagtt ctgtttcccc gtgcgcaagc tgatcgggtc 3240 aaatgacctg ccggactaag atttgaagga ggacgggggg caggctggcc cgatcctagt 3300 catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360 gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420 tagcacgtac attgagaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480 cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540 aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cctgcctggc 3600 ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660 gtcgctgcgc tccctacgcc ccgcCgCttc gcgtcggcct atcgcggccg ctggccgctc 3720 aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780 actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840 aaaacctctg acacatgcag ctcccgctga cggtcacagc ttgtctgtaa gcggatgccg 3900 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020 gattttactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260 ggccgcgttg Ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920 acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980 atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040 ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100 gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160 gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220 ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280 gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340 taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400 cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460 gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520 gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580 atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640 tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700 tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760 tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820 aattcactgt tccttgcatt ctaaaacctt aaatacaaga aaacagcttt ttcaaagttg 5880 ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940 caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000 gtttcaaacc ctgcacctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060 tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120 cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180 tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240 taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300 gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360 ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420 ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480 ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540 ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600 atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660 gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720 cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780 gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggggacgag ccagggatag 6840 cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900 aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960 gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020 gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctcaccaaa tgaaatgaac 7080 ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140 attggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200 cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260 aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttttac 7320 tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380 taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440 cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500 agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560 gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620 tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680 atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740 gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800 gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040 tacgaattcg agctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctt 8100 ggcactggcc gtcgttttac aaCgtcgtga ctgggaaaac cctggcgtta cccaacttaa 8160 tcgccttgca gcacatcccc ctttcgccag ctcgcgtaat agcgaagagg cccgcaccga 8220 tcgcccttcc caacagttgc gcagcctgaa t9gcgaatgc tagagcagct tgagcttgga 8280 tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg 8340 ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa 8400 ggtttatccg ttcgtccatt tgtatgtg 8428 <210> 91 <211> 3438 <212> DNA
<213> Artificial Sequence <220>
<223> pLIT38attBZeo Plasmid <400> 91 tcgaccctct agtcaaggcc ttaagtgagt cgtattacgg actggccgtc gttttacaac 60 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 120 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 180 gcctgaatgg cgaatggcgc ttcgcttggt aataaagccc gcttcggcgg gctttttttt 240 gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 300 tttctaaata cattcaaata tgtatccgct catgagacaa taacccttgt aaatgcttca 360 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 420 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 480 tgctgaagat cagttgggtg cacgagtggg ttacatcaaa ctggatctca acagcggtaa 540 gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 600 gctatgtggc gcggtattat cctgtgttga cgccgggcaa gagcaactcg gtcgccgcat 660 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 720 tggcatgaca gtaagagaat tatgcagtgc tgccatagcc atgagtgata acactgcggc 780 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 840 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 900 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 960 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 1020 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 1080 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 1140 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 1200 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1260 ctcatatata ctttagattg atttaccccg gttgataatc aaaaaagccc caaaaacagg 1320 aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1380 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1440 aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1500 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1560 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1620 = =

aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1680 gaaaggaagg gaagaaagcg aaaggagcgg gcgacagggc gctggcaagt gtagcggtca 1740 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1800 atctaggtga aaatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1860 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1920 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1980 ccggatcaag agctaccaac tttttttccg aaggtaactg gcttcagcag agcgcagata 2040 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2100 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2160 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2220 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2280 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2340 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2400 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2460 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2520 ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2580 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2640 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2700 ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2760 tgctttttta tactaacttg agcgaaatct ggatccatgg ccaagttgac cagtgccgtt 2820 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 2880 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 2940 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 3000 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 3060 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 3120 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta 3180 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 3240 gacgccggct ggatgatcct ccagcgcggg g =ctcatgc tggagttctt cgcccacccc 3300 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 3360 aataaagcat ttttttcact gcattctagt tgtggttttt ccaaactcat caatgtatct 3420 tatcatgtct gtataccg 3438 <210> 92 <211> 10549 <212> DNA
<213> Artificial Sequence <220>
<223> pCambia1302 Plasmid <300>
<308> Genbank #AF234398 <309> 2000-04-24 <400> 92 catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180 gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240 tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300 gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360 agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420 cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480 gcaaaaaaac ggcatcaaag ccaacttcaa gacccgccac aacatgtaag acggcggcgt 540 gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600 agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 660 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 720 atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 900 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020 ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200 ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260 tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 1440 agcacccacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560 cttgaccacc tacgccctgg cgacgttgtg acagtgacca gtctaaaccg cctggcccgc 1620 agcacccgcg acctactgga cattaccgag cgcatccagg aggccggcgc gggcctgcgt 1680 agcctggcag agccgtgggc cgacaccacc acgccggccg gccacatggt gttgaccgtg 1740 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800 gaggccgcca aggcccgagg cgtgaagttt ggcccacgcc ctaccctcac cccggcacag 1860 atcgcgcacg cccgcgaggt gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 1920 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 1980 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100 aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160 tgttCgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220 ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400 taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 2460 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 2520 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580 ccgcgacgtg aaggccatcg ggcggctcga cttcgtagtg atctacggag cgccccaggc 2640 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820 catcggcggt gaggttgccq aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 2880 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940 agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctaaaa ttaaatcaaa 3000 actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 3060 ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180 gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420 caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 3480 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600 aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720 tcatggccgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 3840 tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc ataaaccgat 3900 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960 tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080 tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260 tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380 ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 4440 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560 gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 4620 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740 aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 4800 tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860 ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 4920 gcggacaagc cgcgcacttg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5220 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5280 actcgccgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5820 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120 acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180 agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240 agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300 ttctttttcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360 gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420 gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480 ccaattcggc taagcggttg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540 agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600 cttcatactc ttccgagc.aa aggacgccat cggcctcact catgagcaga ttgctccagc 6660 catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720 tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780 ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840 ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900 ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960 taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020 gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080 gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140 taccctccgc gagatcatcc gtgtttcaaa cctggaagct tagttgccgt tcttccgaat 7200 agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260 cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320 tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380 aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440 tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500 acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 7560 ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620 gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860 gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacgccc tccagaagaa 7980 gatgttggcg accttgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340 tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400 ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg 8460 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640 ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc cgggatctgc 8700 gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760 aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820 atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180 cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240 gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300 gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 936b = =

gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420 aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480 gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660 cttccgactc gtatgttgtg tggaattgtg accggataac aatttcacac aggaaacagc 9720 tatgaccatg attacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag 9780 gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 9840 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 9900 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag 9960 cttgagcttg gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat 10020 tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 10080 cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 10140 ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 10200 acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 10260 tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 10320 ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 10380 gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 10440 tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 10500 tatataagga agttcatttc atttggagag aacacggggg actcttgac 10549 <210> 93 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> CaMV35SpolyA Primer <400> 93 ctgaattaac gccgaattaa ttcgggggat ctg 33 <210> 94 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> CaMV35Spr Primer <400> 94 ctagagcagc ttgccaacat ggtggagca 29 <210> 95 <211> 12592 <212> DNA
<213> Artificial Sequence <220>
<223> pAg2 Plasmid <400> 95 gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta 60 gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag 120 ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc 180 ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg 240 ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg 300 ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc 360 cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc 420 gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc 480 aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca 540 ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattggaaac ccaaagccgt 600 acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt 660 ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac 720 tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct 780 ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg 840 gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc 900 = s' ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 960 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1020 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 1080 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 1140 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 1200 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1260 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1320 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1380 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1440 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1500 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1560 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1620 ttcgctccaa gctgggctgt gggcagcaac ccccagttca gcccgaccgc tgcgccttat 1680 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1740 ccactggtaa cagggttagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1800 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1860 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1920 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1980 atcttttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2040 ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa tattttattt 2100 tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc 2160 cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc 2220 cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt 2280 ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat 2340 catacagctc gcgcgtatct ttaaatggag tgtcttcttc ccagttttcg caatccacat 2400 cggCcagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg 2460 tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct 2520 cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg 2580 cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa 2640 caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg 2700 tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct 2760 tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca 2820 gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct 2880 acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt 2940 ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt 3000 ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac 3060 gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc 3120 ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgcagccat 3180 acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat 3240 tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt 3300 gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga 3360 attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg ttttaggaat 3420 tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg gtttcttata 3480 tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg gaactactca 3540 cacattatta tgaagaaagt cgagtcaaat ctcggtgacg ggcaggaccg gacggggcgg 3600 taccggcagg ctgaagtcca gctgccagaa acccacgtca tgccagttcc cgtgcttgaa 3660 gccggccgcc cgcagcatgc cgcggggggc atatccgagc gcctcgtgca tgcgcacgct 3720 cgggtcgttg ggcagcccga tgacagcgac cacgctcttg aagccctgtg cctccaggga 3780 cttcagcagg tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc ggggggagac 3840 gtacacggtc gactcggccg tccagtcgta ggcgttgcgt gccttccagg ggcccgcgta 3900 ggcgatgccg gcgacctcgc cgtccacctc ggcgacgagc cagggatagc gctcccgcag 3960 acggacgagg tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga agttgaccgt 4020 gcttgtctcg atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg cctcggtggc 4080 acggcggatg tcggccgggc gtcgttctgg gctcatggta gactcgagag agatagattt 4140 gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata 4200 gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 4260 cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 4320 tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 4380 ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 4440 taaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 4500 gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 4560 cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa gacgtggttg 4620 gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 4680 cggcagaggc atcttgaagg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 4740 caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 4800 ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 4860 ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct 4920 S

gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 4980 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5040 gctcactcat taggcacccc aggctttaca ctttatgctt cCggctcgta tgttgtgtgg 5100 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattcga 5160 gccttgacta gagggtcgac ggtatacaga catgataaga tacattgatg agtttggaca 5220 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc 5280 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca 5340 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca 5400 acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag tcctgctcct 5460 cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg 5520 gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga 5580 cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt 5640 tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca 5700 caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact 5760 cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca 5820 tggatccaga tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg caggaattcg 5880 atcgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 5940 attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 6000 atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 6060 tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 6120 cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 6180 gtggattgat gtgataacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 6240 gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 6300 aaccttctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 6360 gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 6420 tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 6480 gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 6540 gatgacgcac aatcccacta tccttcgcaa gaccttcctc tatataagga agttcatttc 6600 atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct ctctcgagct 6660 ttcgcagatc cgggggggca atgagatatg aaaaagcctg aactcacc9c gacgtctgtc 6720 gagaagtttc tgatCgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 6780 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 6840 agctgggccg atggtttcta caaagatcgt tatgtttatc ggcacttttc atcggccgcg 6900 ctccc attc cggaagtgct tgacattggg gagtttagcg agagcctgac ctattgcatc 6960 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 7020 ctacaaccgg tcgcggaggc tatggatgcg atcgctgcgg ccgatcttag ccagacgagc 7080 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 7140 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 7200 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 7260 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 7320 acagtggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 7380 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 7440 aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt 7500 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 7560 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 7620 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcggcgatag tggaaaccga 7680 cgccccagca ctcgtccgag ggaaaagaaa tagagtagat gccgaccgga tctgtcgatc 7740 gacaagctgg agtttctcca taataatgtg tgagtagttc ccagataagg gaattagggt 7800 tcctataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt 7860 tgtaaaatac ttctatcaat aaaatttcta attcctaaaa caaaaatcca gtactaaaat 7920 ccagatcccc cgaattaatt cggcgttaat tcagatcaag cttggcactg gccgtcgttt 7980 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 8040 cccctttcgc cagctggcgt aatagcgaag aggcgcgcac cgatcgccct tcccaacagt 8100 tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt gtcgtttccc 6160 gccttcagtt tggggatcct ctagactgaa ggcgggaaac gacaatctga tcatgagcgg 8220 agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc gttttacgtt 8280 tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc tggagtttaa 8340 tgaggtaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 8400 atcatccagc aaatatttct tgtcaaaaat gctccactga cgttccataa attcccctcg 8460 gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg gatctcgaga 8520 atcgaattcc cgcggccgcc atggtacatc tgactagtaa aggagaagaa cttttcactg 8580 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca 8640 gtggagaggg taaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta 8700 ctggaaaact acctgttccg tggccaacac ttgtcactac tttctcttat ggtgttcaat 8760 gcttttcaag atacccagat catatgaagc ggcacgactt cttcaagagc gccatgcctg 8820 agggatacgt gcaggagagg accatcttct tcaaggacga cgggaactac aagacacgtg 8880 ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat cgagcttaag ggaatcgatt 8940 tcaaggagga cggaaacatc ctcggccaca agttggaata caactacaac tcccacaacg 9000 tatacatcat ggccggcaag caaaagaacg gcatcaaagc caacttcaag acccgccaca 9060 acatcgaaga cggcggcgtg caactcgctg atcattatca acaaaatact ccaattggcg 9120 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag 9180 atcccaacga aaagagagac cacatggccc ttcttgagtt tgtaacagct gctgggatta 9240 cacatggcat ggatgaacta tacaaagcta gccaccacca ccaccaccac gtgtgaattg 9300 gtgaccagct cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg 9360 aatcctgttg ccggtctttc gatgattatc atataatttc tgttgaatta cgttaagcat 9420 gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc 9480 ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa 9540 ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat taaactatca gtgtttgaca 9600 gtgtatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac ggatatttaa 9660 aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc 9720 ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg 9780 acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca 9840 ggctgccgcc ctgccctttt cctggcgttt ttttctcgcg tgttttagtc gcataaagta 9900 gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct 9960 gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact 10020 gcaagcggcc ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg 10080 cccggagctg gccaggatgc ttgaccacct acgccctggc gacgttgtga cagtgaccag 10140 gctagaccgc ctggcccgca gcacccgcga cctactggac attgccgagc gcatccagga 10200 ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc gacaccacca cgccggccgg 10260 ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc taatcatcga 10320 ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc 10380 taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac 10440 cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg accctgtacc gcgcacttga 10500 gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc 10560 attgaccgag gccgacgccc tggcggccgc cgagaatgaa cgccaagagg aacaagcatg 10620 aaaccgcacc aggacggcca ggacgaaccg tttttcatta ccgaagagat cgaggcggag 10680 atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat 10740 gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct ggccggccag cttggccgct 10800 gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt 10860 catgcggtcg ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa ggggaacgca 10920 tgaaggttat cgctgtactt aaccagaaag gcgggtcagg caagacgacc atcgcaaccc 10980 atctagcccg cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat tccgatcccc 11040 agggcagtgc ccgcgattgg gcggccgtgc gggaagatca accgctaacc gttgtcggca 11100 tcgaccgccc gacgattgac cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga 11160 tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg 11220 tgctgattcc ggtgcagcca agcccttacg acatatgggc caccgccgac ctggtggagc 11280 tggttaagca gcgcattgag gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc 11340 gggcgatcaa aggcacgcgc atcggcggtg aggttgccga ggcgctggcc gggtacgagc 11400 tgcccattct tgagtcccgt atcacgcagc gcgtgagcta cccaggcact gccgccgccg 11460 gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg 11520 ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag 11580 cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg caacgttggc 11640 cagcctggca gacacgccag ccatgaagcg ggtcaacttt cagttgccgg cggaggatca 11700 caccaagctg aagatgtacg cggtacgcca aggcaagacc attaccgagc tgctatctga 11760 atacatcgcg cagctaccag agtaaatgag caaatgaata aatgagtaga tgaattttag 11820 cggctaaagg aggcggcatg gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc 11880 ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc tgccggccct 11940 gcaatggcac tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa accatccggc 12000 ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag 12060 gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc 12120 gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg 12180 aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc 12240 acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga 12300 cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg 12360 ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta 12420 accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt 12480 ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac 12540 gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca gc 12592 <210> 96 c211> 3357 <212> DNA
<213> Artificial Sequence <220>
<223> pGEMEasyNOS Plasmid <400> 96 tatcactagt gaattcgcgg ccgcctgcag gtcgaccata tgggagagct cccaacgcgt 60 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca 120 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 180 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 240 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 300 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 360 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 420 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtaagcaaa aggccagcaa 480 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 540 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 600 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 660 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcggtttc tcatagctca 720 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 780 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 840 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 900 tatgtaggcg gtgctacaga gttcttgaag tggttgccta actacggcaa cactagaaga 960 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 1020 tcgtgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 1080 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 1140 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 1200 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 1260 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1320 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1380 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 1440 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 1500 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 1560 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 1620 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 1680 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 1740 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 1800 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 1860 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 1920 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggagaaaatt ctcaaggatc 1980 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 2040 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 2100 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 2160 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 2220 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga 2280 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt 2340 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 2400 atcggcaaaa tcccttataa attaaaagaa tagaccgaga tagggttgag tgttgttcca 2460 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 2520 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 2580 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 2640 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 2700 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 2760 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 2820 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 2880 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt 2940 aatacgactc actatagggc gaattgggcc cgacgtcgca tgctcccggc cgccatggcg 3000 gccgcgggaa ttcgattctc gagatccggt gcagattatt tggattgaga gtgaatatga 3060 gactctaatt ggataccgag gggaatttat ggaacgtcag tggagcattt ttgacaagaa 3120 atatttgcta gctgatagtg accttaggcg acttttgaac gcgcaataat ggtttctgac 3180 gtatgtgctt agctcattta actccagaaa cccgcggctg agtggctcct tcaacgttgc 3240 ggttctgtca gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg 3300 tgactccctt aattctccgc tcatgatcag attgtcgttt cccgccttca gtctaga 3357 <210> 97 <211> 10122 <212> DNA
<213> Artificial Sequence <220>

<223> p1302NOS Plasmid <400> 97 catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180 gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240 tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300 gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360 agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420 catcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480 gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag acggcggcgt 540 gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600 agacaaccat tacctgtcca cacaatttgc cctttcgaaa gatcccaacg aaaagagaga 660 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggta tggatgaact 720 atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840 cgatgattat catataattt cttttgaatt acgttaagca tgtaataattlaacatgtaat 900 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960 acgcgataaa aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020 ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200 ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260 tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggata tgcccgcgtc 1440 agcacccacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560 cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 1620 agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 1680 agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 1740 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800 gaggccgcca aggcccgagg cgtgaagttt ggcccacgcc ctaccctcac cccggcacag 1860 atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgagaaaga ggcggctgca 1920 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 1980 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100 aggcgcaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160 tgttcgagcc gcccgcgCac gtCtcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220 ctgatgccaa gctggCggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400 taaccagaaa ggcgtgtcag gcaagac ac catcgcaacc catctagccc gcgccctgca 2460 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcaattg 2520 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580 ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 2640 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820 catcggcggt gaggttgccg aggctctcgc cgggtacgag ctgcccattc ttgagtcccg 2880 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940 agaacccgag ggcgacgctg cccgcgaggt ccaggcggc9 gccgctgaaa ttaaatcaaa 3000 actcatttga gttaaggagg taaagagaaa atgagcaaaa gcaaaaaaac gctaagtgcc 3060 gcccgtgcga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180 ggcgtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420 caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggcacaa atcggcgcgg 3480 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600 aatgccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720 tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggtccgc atggccagtg 3840 =

tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 3900 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960 tcaagttctg ctggcaagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080 tggggacgtt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260 tccgccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380 ccgtgcgcaa gctgatcggg tcaaatgacc tgccgacgta cgatttgaag gaggaggcgg 4440 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560 gaaaaggttt ctttcctgtg gatagcacgt acattggcaa cccaaagccg tacattggga 4620 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740 aaactcttaa aacccgcctg gcctgtgcat aaCtgtctgg ccagcgcaca gccgaagagc 4800 tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860 ctatcagggc cgctggccgc tcaaaaatgg cttccctacg gccaggcaat ctaccagggc 4920 gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5220 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccccttcc tcgctcactg 5280 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400 aaaaggccag gaaccggaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagaacga 5820 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120 acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180 agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240 agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300 ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360 gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420 gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480 ccatttcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540 agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600 cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 6660 catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720 tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780 ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840 ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900 ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960 taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020 gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080 gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140 taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 7200 agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260 cggactgatg ggttgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320 tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380 aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440 tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500 acaaatacaa atacatacta agggtttctt atatgctcaa cacatgtaag aaaccctata 7560 ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620 gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860 = ., gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa 7980 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340 tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400 ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaattcg 8460 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580 ttcgccctcc gagagctaca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640 ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc ccggatctgc 8700 gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760 aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820 atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180 cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240 gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300 gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 9360 gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420 aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480 gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9720 tatgaccatg attacgaatt cgagctcggt acccggggat cctctagact gaaggcggga 9780 aacgacaatc tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga 9840 cgcgggacaa gccgttttac gtttggaact gacagaaccg caacgttgaa ggagccactc 9900 agccggggat ttctggagtt taatgagcta agcacatacg tcagaaacca ttattgcgcg.9960 ttcaaaagtc gcctaaggtc actatcagct agcaaatatt tcttgtcaaa aatgctccac 10020 tgacgttcca taaattcccc tcggtatcca attagagtct catattcact ctcaatccaa 10080 ataatctgca ccggatctcg agaatcgaat tcccgcggcc gc 10122 <210> 98 <211> 621 <212> DNA
<213> Artificial Sequence <220>
<223> N. tabacum rDNA intergnic spacer (IGS) sequence <300>
<308> Genbank #Y08422 <309> 1997-10-31 <400> 98 gtgctagcca atgtttaaca agatgtcaag cacaatgaat gttggtggtt ggtggtcgtg 60 gctcgcggtg gtggaaaatt gcggtggttc gagcggtagt gatcggcgat ggttggtgtt 120 tgcagcggtg tttgatatcg gaatcactta tggtggttgt cacaatggag gtgcgtcatg 180 gttattggtg gttggtcatc tatatatttt tataataata ttaagtattt tacctatttt 240 ttacatattt tttattaaat ttatgcattg tttgtatttt taaatagttt ttatcgtact 300 tgttttataa aatattttat tattttatgt gttatattat tacttgatgt attggaaatt 360 ttctccattg ttttttctat atttataata attttcttat ttttttttgt tttattatgt 420 attttttcgt tttataataa atatttatta aaaaaaatat tatttttgta aaatatatca 480 tttacaatgt ttaaaagtca tttgtgaata tattagctaa gttgtacttc tttttgtgca 540 tttggtgttg tacatgtcta ttatgattct ctggccaaaa catgtctact cctgtcactt 600 gggttttttt ttttaagaca t 621 <210> 99 <211> 25 <212> DNA

<213> Artificial Sequence <220>
<223> NTIGS-F1 Primer <400> 99 gtgctagcca atgtttaaca agatg 25 <210> 100 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> NTIGS-R1 Primer <400> 100 atgtcttaaa aaaaaaaacc caagtgac 28 <210> 101 <211> 233 <212> DNA
<213> Mus Musculus <300>
<308> Genbank #V00846 <309> 1989-07-06 <400> 101 gacctggaat atggcgagaa aactgaaaat cacggaaaat gagaaataca cactttagga 60 cgtgaaatat ggcgaggaaa actgaaaaag gtggaaaatt tagaaatgtc cactgtagga 120 cgtggaatat ggcaagaaaa ctgaaaatca tggaaaatga gaaacatcca cttgacgact 180 tggaaaatga cgaaatcact aaaaaacgtg aaaaatgaga aatgcacact gaa 233 <210> 102 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> MSAT-F1 Primer <400> 102 aataccgcgg aagcttgacc tggaatatcg c 31 <210> 103 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> MSAT-Ri Primer <400> 103 ataaccgcgg agtccttcag tgtgcat 27 <210> 104 <211> 277 <212> DNA
<213> Artificial Sequence <220>
<223> Nopaline Synthase Promoter Sequence <300>
<308> Genbank #U09365 <309> 1997-10-17 <400> 104 gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60 tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120 aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180 attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240 gcgcgcggtg tcatctatgt tactagatcg ggaattc 277 <210> 105 <211> 1812 <212> DNA
<213> Escherichia coli <220>
<221> CDS
<222> (1)...(1812) <223> Beta-Glucuronidase <300>
<308> Genbank #S69414 <309> 1994-09-23 <400> 105 atg tta cgt cct gta gaa acc cca acc cgt gaa atc aaa aaa ctc gac 48 Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp ggc ctg tgg gca ttc agt ctg gat cgc gaa aac tgt gga att gat cag 96 Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln cgt tgg tgg gaa agc gcg tta caa gaa agc cgg gca att get gtg cca 144 Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro ggc agt ttt aac gat cag ttc gcc gat gca gat att cgt aat tat gcg 192 Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala ggc aac gtc tgg tat cag cgc gaa gtc ttt ata ccg aaa ggt tgg gca 240 Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala ggc cag cgt atc gtg ctg cgt ttc gat gcg gtc act cat tac ggc aaa 288 Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys gtg tgg gtc aat aat cag gaa gtg atg gag cat cag ggc ggc tat acg 336 Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr cca ttt gaa gcc gat gtc acg ccg tat gtt att gcc ggg aaa agt gta 384 Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val cgt atc acc gtt tgt gtg aac aac gaa ctg aac tgg cag act atc ccg 432 Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro ccg gga atg gtg att acc gac gaa aac ggc aag aaa aag cag tct tac 480 Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr ttc cat gat ttc ttt aac tat gcc gga atc cat cgc agc gta atg ctc 526 Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu tac acc acg ccg aac acc tgg gtg gac gat atc acc gtg gtg acg cat 576 = =

Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His gtc gcg caa gac tgt aac cac gcg tct gtt gac tgg cag gtg gtg gcc 624 Val Ala Gln Asp Cys Asn His Ala Ser Val Asp 'Trp Gln Val Val Ala aat ggt gat gtc agc gtt gaa ctg cgt gat gcg gat caa cag gtg gtt 672 Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val gca act gga caa ggc act agc ggg act ttg caa gtg gtg aat ccg cac 720 Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His ctc tgg can ccg ggt gaa ggt tat ctc tat gaa ctg tgc gtc aca gcc 768 Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala aaa agc cag aca gag tgt gat atc tac ccg ctt cgc gtc ggc atc cgg 816 Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg tca gtg gca gtg aag ggc gaa cag ttc ctg att aac cac aaa ccg ttc 864 Ser Val Ala Val Lys Gly Glu Gin Phe Leu Ile Asn His Lys Pro Phe tac ttt act ggc ttt ggt cgt cat gaa gat gcg gac ttg cgt ggc aaa 912 Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys gga ttc gat aac gtg ctg atg gtg cac gac cac gca tt.a atg gac tgg 960 Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp att ggg gcc aac tcc tac cgt acc tcg cat tac cct tac get gaa gag 1008 Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu atg ctc gac tgg gca gat gaa cat ggc atc gtg gtg att gat gaa act 1056 Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr get get gtc ggc ttt aac ctc tct tta ggc att ggt ttc gaa gcg ggc 1104 Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly aac aag ccg aaa gaa ctg tac agc gaa gag gca gtc aac ggg gaa act 1152 Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr cag caa gcg cac tta cag gcg att aaa gag ctg ata gcg cgt gac aaa 1200 Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys aac cac cca agc gtg gtg atg tgg agt att gcc aac gaa ccg gat acc 1248 Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr cgt ccg caa ggt gca cgg gaa tat ttc gcg cca ctg gcg gaa gca acg 1296 Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr cgt aaa ctc gac ccg acg cgt ccg atc acc tgc gtc aat gta atg ttc 1344 Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe = S

tgc gac get cac acc gat acc atc agc gat ctc ttt gat gtg ctg tgc 1392 Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys ctg aac cgt tat tac gga tgg tat gtc caa agc ggc gat ttg gaa acg 1440 Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr gca gag aag gta ctg gaa aaa gaa ctt ctg gcc tgg cag gag aaa ctg 1488 Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gin Glu Lys Leu cat cag ccg att atc atc acc gaa tac ggc gtg gat acg tta gcc ggg 1536 His Gin Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly ctg cac tca atg tac acc gac atg tgg agt gaa gag tat cag tgt gca 1584 Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala tgg ctg gat atg tat cac cgc gtc ttt gat cgc gtc agc gcc gtc gtc 1632 Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val ggt gaa cag gta tgg aat ttc gcc gat ttt gcg acc tcg caa ggc ata 1680 Gly Glu Gin Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile ttg cgc gtt ggc ggt aac aag aaa ggg atc ttc act cgc gac cgc aaa 1728 Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys ccg aag tcg gcg get ttt ctg ctg caa aaa cgc tgg act ggc atg aac 1776 Pro Lys Ser Ala Ala Phe Leu Leu Gin Lys Arg Trp Thr Gly Met Asn ttc ggt gaa aaa ccg cag cag gga ggc aaa caa tga 1812 Phe Gly Glu Lys Pro Gin Gln Gly Gly Lys Gin <210> 106 <211> 603 <212> PRT
<213> Escherichia coli <300>
<308> Genbank $#S69414 <309> 1994-09-23 <400> 106 Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gin Glu Ser Arg Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gin Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gin Arg Glu Val Phe Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gin Glu Val Met Glu His Gin Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val =

Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr Gln Gin Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val Gly Glu Gin Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile 545 550 555 560.
Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln <210> 107 <211> 277 <212> DNA
<213> Artificial Sequence = =

<220>
<223> Nopaline Synthase Terminator Sequence <300>
<308> U09365 <309> 1995-10-17 <400> 107 gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60 tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120 aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180 attatacatt taatacgcca tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240 gcgcgcggtg tcatctatgt tactagatcg ggaattc 277 <210> 108 <211> 3451 .<212> DNA
<213> Artificial Sequence <220>
<223> HindIll Fragment containing the beta-glucuronidase coding sequence, the rDNA intergenic spacer, and the Mastl sequence <400> 108 aagcttgacc tggaatatcg cgagtaaact gaaaatcacg gaaaatgaga aatacacact 60 ttaggacgtg aaatatggcg aggaaaactg aaaaaggtgg aaaatttaga aatgtccact 120 gtaggacgtg gaatatggca agaaaactga aaatcatgga aaatgagaaa catccacttg 180 acgacttgaa aaatgacgaa atcactaaaa aacgtgaaaa atgagaaatg cacactgaag 240 gactccgcgg gaattcgatt gtgctagcca atgtttaaca agatgtcaag cacaatgaat 300 gttggtggtt ggtggtcgtg gctggcggtg gtggaaaatt gcggtggttc gagcggtagt 360 gatcggcgat ggttggtgtt tgcagcggtg tttgatatcg gaatcactta tggtggttgt 420 cacaatggag gtgcgtcatg gttattggtg gttggtcatc tatatatttt tataataata 480 ttaagtattt tacctatttt ttacatattt tttattaaat ttatgcattg tttgtatttt 540 taaatagttt ttatcgtact tgttttataa aatattttat tattttatgt gttatattat 600 tacttgatgt attggaaatt ttctccattg ttttttctat atttataata attttcttat 660 ttttttttgt tttattatgt attttttcgt tttataataa atatttatta aaaaaaatat 720 tatttttgta aaatatatca tttacaatgt ttaaaagtca tttgtgaata tattagctaa 780 gttgtacttc tttttgtgca tttggtgttg tacatgtcta ttatgattct ctggccaaaa 840 catgtctact cctgtcactt gggttttttt ttttaagaca taatcactag tgattatatc 900 tagactgaag gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgtta 960 tgacccccgc cgatgacgcg ggacaagccg ttttacgttt ggaactgaca gaaccgcaac 1020 gttgaaggag ccactcagcc gcgggtttct ggagtttaat gagctaagca catacgtcag 1080 aaaccattat tgcgcgttca aaagtcgcct aaggtcacta tcagctagca aatatttctt 1140 gtcaaaaatg ctccactgac gttccataaa ttcccctcgg tatccaatta gagtctcata 1200 ttcactctca atccaaataa tctgcaccgg atctcgagat cgaattcccg cggccgcgaa 1260 ttcactagtg gatccccggg tacggtcagt cccttatgtt acgtcctgta gaaaccccaa 1320 cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg 1380 gaattgagca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag 1440 gcagttttaa cgatcagttc gccgatgtag atattcgtaa ttatgtgggc aacgtctggt 1500 atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg 1560 atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg 1620 gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac 1680 gtatcacagt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 1740 ttaccgacga aaacggcaag aaaaagcagt cttactttca tgatttcttt aactacgccg 1800 ggatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 1860 tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 1920 atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 1980 gcaccagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 2040 tctatgaact gtacgtcaca gccaaaagcc agacagagtg tgatatctac ccgctgcgcg 2100 tcggcatccg gtcagtggca gtgaagggcg aacagttcct gatcaaccac aaaccgttct 2160 actttactgg ctttggccgt catgaagatg cggatttgcg cggcaaagga ttcgataacg 2220 tgctgatggt gcacgatcac gcattaatgg actggattgg ggccaactcc taccgtacct 2280 cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga 2340 ttgatgaaac tgcagctgtc ggctttaacc tctctttagg cattggtttc gaagcgggca 2400 acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caggcgcact 2460 tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 2520 gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 2580 cggaagcaac gcgtaaactc gatccgacgc gtccgatcac ctgcgtcaat gtaatgttct 2640 gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 2700 acggttggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 2760 ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 2820 cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 2880 ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 2940 ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaagg 3000 ggatcttcac ccgcgaccgc aaaccgaagt cgccggcttt tctgctgcaa aaacgctgga 3060 ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaatgaatc aacaactctc 3120 ctggcgcacc atcgtcggct acagcctcgg gaattgcgta ccgagctcga atttccccga 3180 tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 3240 gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 3300 gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 3360 gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 3420 gttactagat cgggaattcg atatcaagct t 3451 <210> 109 <211> 14627 <212> DNA
<213> Artificial Sequence <220>
<223> pA911a Plasmid <400> 109 catgcgaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60 atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120 agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180 gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240 agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300 ccaaccaacg ggccgaactg cactcggccg gctgcaccaa gctgttttcc gagaagatca 360 ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420 acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccacgac ctactggaca 480 ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540 acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600 agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660 tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720 tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780 ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840 gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900 gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960 cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020 ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080 gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140 tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200 aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260 aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320 ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380 ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440 cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500 atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560 accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620 gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680 gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740 ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800 cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860 aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920 gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980 agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa gggaagacca 2040 ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100 atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160 accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220 tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280 cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340 gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400 tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460 cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520 gatgctctat gacgtgggga cccgcgatag tcgcagcatc atggacgtgg cggttttcag 2580 tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640 cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtagt 2700 gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760 gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820 tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880 tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940 agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000 gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060 gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120 gccacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180 cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240 aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300 catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360 gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420 tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480 cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540 aggcgctttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600 ctgtgcataa ctgtctggcc agcgcacagc caaacagctg caaaaagccc ctacccttcg 3660 gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720 aaaaatggct ggcctacggc caggcaatgt accagggcgc ggacaagccg cgccgtcgcc 3780 actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920 acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980 atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040 ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100 gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160 gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220 ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280 gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340 taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400 cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460 gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520 gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580 atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640 tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700 tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760 tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820 aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880 ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940 caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000 gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060 tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120 cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180 tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240 taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300 gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360 ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420 ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480 . =

ggacggggcg gtaccggcag gctgaagtcc agcttccaga aacccacgtc atgccagttc 6540 ccgtgcttga agccggccgc ccgcagcatg ccgggggggg catatccgag cgcctcgtgc 6600 atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660 gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720 cggggggaga cgtactcggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780 gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840 cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900 aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960 gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020 gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080 ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cctttatctc 7140 agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200 cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260 aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320 tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380 taccctttgt tgaaaagtct caatacccct ttggtcttct gagactgtat ctttaatatt 7440 cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500 agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560 gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620 tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680 atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740 gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800 gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980 atgttgtgtg gaattgtgag cggataacaa tttcaaacag gaaacagcta tgaccatgat 8040 tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 8340 gtcctgctcc tcggccacga agtgcacgca gtttccggcc gggtcgcgca gggcgaactc 8400 ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460 cgtggacacg acctccgacc actcggcgta caactcgtcc aggccgcgca cccacaccca 8520 ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580 gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640 ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700 caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760 gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820 accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880 attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940 aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 9060 cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 9120 agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180 taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240 cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9300 ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360 tgaaaaaaaa agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420 ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480 aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 9540 tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 9720 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9780 catcggccgc gctcccgatt ccggaagtcc ttgacattgg ggagtttagc gagagcctga 9840 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9900 tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 10260 aggtcgccaa catctttttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320 acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440 gggcgcaggg ttgatgcaac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500 = =

aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560 gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620 atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680 ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740 gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800 agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttgacctg 10860 gaatatcgcg agtaaactga aaatcacgga aaatgagaaa tacacacttt aggacgtgaa 10920 atatggcgag gaaaactgaa aaaggtggaa aatttagaaa tgtcaactgt aggacgtgga 10980 atatggcaag aaaactgaaa atcatggaaa atgagaaaca tccacttgac gacttgaaaa 11040 atgacgaaat cactaaaaaa cgtgaaaaat gagaaatgca cactgaagga ctccgcggga 11100 attcgattgt gctagccaat gtttaacaag atgtcaagca caatgaatgt tggtggttgg 11160 tggtcgtggc tggcggtggt ggaaaattgc ggtggttcga gcggtagtga tcggcgatgg 11220 ttggtgtttg cagcggtgtt tgatatcgga atcacttatg gtggttgtca caatggaggt 11280 gcgtcatggt tattggtggt tggtcatcta tatattttta taataatatt aagtatttta 11340 cctatttttt acatattttt tattaaattt atgcattgtt tgtattttta aatagttttt 11400 atcgtacttg ttttataaaa tattttatta ttttatgtgt tatattatta cttgatgtat 11460 tggaaatttt ctccattgtt ttttctatat ttataataat tttcttattt ttttttgttt 11520 tattatgtat tttttcgttt tataataaat atttattaaa aaaaatatta tttttgtaaa 11580 atatatcatt tacaatgttt aaaagtcatt tgtgaatata ttagctaagt tgtacttctt 11640 tttgtgcatt tggtgttgta catgtctatt atgattctct ggccaaaaca tgtctactcc 11700 tgtcacttgg gttttttttt ttaagacata atcactagtg attatatcta gactgaaggc 11760 gggaaacgac aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg 11820 atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc 11880 actcagccgc gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg 11940 cgcgttcaaa agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct 12000 ccactgacgt tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat 12060 ccaaataatc tgcaccggat ctcgagatcg aattcccgcg gccgcgaatt cactagtgga 12120 tccccgggta cggtcagtcc cttatgttac gtcctgtaga aaccccaacc cgtgaaatca 12180 aaaaactcga cggcctgtgg gcattcagtc tggatcgcga aaactgtgga attgagcagc 12240 gttggtggga aagcgcgtta caagaaagcc gggcaattgc tgtgccaggc agttttaacg 12300 atcagttcgc cgatgcagat attcgtaatt atgtgggcaa cgtctggtat cagcgcgaag 12360 tctttatacc gaaaggttgg gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc 12420 attacggcaa agtgtgggtc aataatcagg aagtgatgga gcatcagggc ggctatacgc 12480 catttgaagc cgatgtcacg ccgtatgtta ttggcgggaa aagtgtacgt atcacagttt 12540 gtgtgaacaa cgaactgaac tggcagacta tcccgccggg aatggtgatt accgacgaaa 12600 aggccaagaa aaagcagtct tacttccatg atttctttaa ctacgccggg atccatcgca 12660 gcgtaatgct ctacaccacg ccgaacacct gggtggacga tatcaccgtg gtgacgcatg 12720 tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt ggtggccaat ggtgatgtca 12780 gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc accagcggga 12840 ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc tatgaactgt 12900 acgtcacagc caaaagccag acagagtgtg atatctaccc gctgcgcgtc ggcatccggt 12960 cagtggcagt gaagggcgaa cagttcctga tcaaccacaa accgttctac tttactggct 13020 ttggccgtca tgaagatgcg gatttgcgcg gcaaaggatt cgataacgtg ctgatggtgc 13080 acgatcacgc attaatggac tggattgggg ccaactccta ccgtacctcg cattaccctt 13140 acgctgaaga gatgctcgac tgggcagatg aacatggcat cgtggtgatt gatgaaactg 13200 cagctgtcgg ctttaacctc tctttaggca ttggtttcga agcgggcaac aagccgaaag 13260 aactgtacag cgaagaggca gtcaacgggg aaactcagca ggcgcactta caggcgatta 13320 aagagctgat agctcgtgac aaaaaccacc caagcgtggt gatgtggagt attgccaacg 13380 aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc gccactggcg gaagcaacgc 13440 gtaaactcga tccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 13500 ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggttggtatg 13560 tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 13620 aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 13680 tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 13740 atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 13800 attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaagggg atcttcaccc 13860 gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact gccatgaact 13920 tcggtgaaaa accgcagcag ggaggcaaac aatgaatcaa caactctcct ggcgcaccat 13980 cgtcggctac agcctcggga attgcgtacc gagctcgaat ttccccgatc gttcaaacat 14040 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 14100 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 14160 gatatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 14220 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 14280 ggaattcgat atcaagcttg gcactagccg tcgttttaca acgtcgtgac tgggaaaacc 14340 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 14400 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct 14460 agagcagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt 14520 S

ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataacggat 14580 atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtg 14627 <210> 110 <211> 9080 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeo(6XIS4)2eGFP Plasmid <400> 110 cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc cagcccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420 agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660 acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900 atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggatccgc tccggtccgg 1020 cgctcccccc gcatccccga gccggcagcg ttcggggaca gcccgggcac ggggaaagtg 1080 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140 ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200 aattacgtcc ctcccccggt agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380 ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgcccgggt 1500 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620 tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740 gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800 aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860 cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920 tagttcatag cccatatatg gagttcctcg ttacataact tacggtaaat ggcccggctg 1980 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040 cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220 acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280 actctcccca tccccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340 ttttgtgcag cgatgggggc ggggggtggg ggggcgcgcg ccaggcgggg cggggggggg 2400 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaaccgc 2520 gcggcgggc ggagtcgctg cgttgccttc gccccgtgcc ccgctcaggg ccgcctcgcg 2580 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700 gcgtgaaagc cttaaagggc tccgggaagg ccctttgtgc ggggggtagc ggctcggggg 2760 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtcccg cccgcgctgc ccggcggctg 2820 tgagcgctgc gggcgcggcg cggggctttg tcggatccgc gtgtgcgcga ggggagcgcg 2880 gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120 ggggcggggc cccctcggcc cggggagggc tcggggaagg gccgcggcgg ccccggggcg 3180 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240 gggcacgggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300 caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420 cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540 tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3720 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4200 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 4380 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560 atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620 caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860 cccaagcttg catgcctgca ggtcgactct agtggatccc ccgccccgta tcccccaggt 4920 gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc 4980 cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc 5040 ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc 5100 cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggccccg tatcccccag 5160 gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct 5220 ttcccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga 5280 ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca 5340 tccctggggg ctttgggggg gggctgtccc cgtgagcgga tccgcggccc cgtatccccc 5400 aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac 5460 cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg 5520 gaccggagcg gacccccggg cggctggctg ctgcccccta gcgggggagg gacgtaatta 5580 catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc cccgtatccc 5640 ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc 5700 actgtccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc 5760 cgacccgcag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat 5820 tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg gccccgtatc 5880 ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg 5940 ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc 6000 gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta 6060 attacatccc tgggggcttt gggggggggc tgtccccgtg agcgaatccg cggccccgta 6120 tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg 6180 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga 6240 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg 6300 taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggggctg 6360 caggaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 6420 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 6480 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 6540 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 6600 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 6660 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 6720 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 6780 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 6840 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 6900 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6960 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7020 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7080 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7140 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7200 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 7260 cttcggaaaa agagttggta gctctttatc cggcaaacaa accaccgctg gtagcggtgg 7320 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7380 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7440 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7500 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7560 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7620 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 7680 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 7740 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 7800 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 7860 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 7920 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7980 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8040 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8100 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccggcgtcaatacg 8160 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8220 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8280 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8340 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8400 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8460 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8520 agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa gcgggcttta ttaccaagcg 8580 aagcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 8640 tcgctattac gccagctggc gaaagggcga tgtgctgcaa ggcgattaag ttgggtaacg 8700 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtccgtaata cgactcactt 8760 aaggccttga ctagagggtc gacggtatac agacatgata agatacattg atgagtttgg 8820 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 8880 tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca 8940 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc 9000 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tagcacgtgt cagtcctgct 9060 cctcggccac gaagtgcacg 9080 <210> 111 <211> 4223 <212> DNA
<213> Artificial Sequence <220>
<223> pLIT38attBBSRpolyAlO Plasmid <400> 111 gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300 gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020 ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080 aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200 aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380 aatgggaacc ctaaagggag cccccgattt agagcttgac ggagaaagcg aacgtggcga 1440 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620 S

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280 ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460 ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520 tgctttttta tactaacttg agcgaaatct ggatcaccat gaaaacattt aacatttctc 2580 aacaagatct agaattagta gaagtagcga cagagaagat tacaatgctt tatgaggata 2640 ataaacatca tgtgggagcg gcaattcgta cgaaaacagg agaaatcatt tcggcagtac 2700 atattgaagc gtatatagga cgagtaactg tttgtgcaga agccattgcg attggtagtg 2760 cagtttcgaa tggacaaaag gattttgaca cgattgtagc tgttagacac ccttattctg 2820 acgaagtaga tagaagtatt cgagtggtaa gtccttgtgg tatgtgtagg gatttgtttt 2880 cagactatgc accagattgt tttgtgttaa tagaaatgaa tggcaagtta gtcaaaacta 2940 cgattgaaga actcattcca ctcaaatata cccgaaatta aaagttttac cataccaagc 3000 ttggctgctg cctgaggctg gacgacctcg cggagttcta ccggcagtgc aaatccgtcg 3060 gcatccagga aaccagcagc ggctatccgc gcatccatgc ccccgaactg caggagtggg 3120 gaggcacgat ggccgctttg gtccggatct ttgtgaagga accttacttc tgtggtgtga 3180 cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa 3240 gtgtataatg tgttaaacta ctaattctaa ttgtttgtgt attttagatt ccaacctatg 3300 gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag 3360 aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa 3420 aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga 3480 gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa 3540 aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc 3600 ataacagtta taatcataac atactgtttt ttcttactcc atacaggcat agagtgtctg 3660 ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta 3720 ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt 3780 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 3840 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 3900 aatagcatca caaatttcac aaataaagat ccacgaattc gctagcttcg gccgtgacgc 3960 gtctccggat gtacaggcat gcgtcgaccc tctagtcaag gccttaagtg agtcgtatta 4020 cggactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 4080 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 4140 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcttcgctt ggtaataaag 4200 cccgcttcgg cgggcttttt ttt 4223 <210> 112 <211> 5855 <212> DNA
<213> Artificial Sequence <220>
<223> pCX-LamTntR Plasmid <400> 112 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtgat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagctaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccccc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 S

gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tggggggttg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcgggggtga cggggcaggg cggggttcgg cttctggcgt gtgagcgccg 1620 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740 gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800 acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atcgcaatca 1860 ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920 cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980 tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040 caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaattgcgg 2100 caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160 cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220 ctgccactcg cgcagcaaaa tctagagtaa ggagatcaag aattactgct gacgaatacc 2280 tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340 ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400 atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460 tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520 ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580 caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640 cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700 ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760 gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 2880 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060 gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180 ggttagattt tttttatatt ttgttgtgtg ttattttttt ctttaacatc cctaaaattt 3240 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300 gtccctcttc tctaatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600 tccgcccatt ctccagccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3660 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720 tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggtaccg 4080 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260 gtccgccttt ctcccttcgg gaagcgtggc gctttcttaa tgctcacgct gtaggtatct 4320 cagttcggtg taggtcgttc ggtcaaagct gggctgtgtg cacgaacccc Ccgttcagcc 4380 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5520 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640 cagcgtttct gggtgaccaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820 ggtttcgcgc acatttcccc gaaaagtgcc acctg 5855 <210> 113 <211> 4346 <212> DNA
<213> Artificial Sequence <220>
<223> pSV40-193AttpsensePur Plasmid <400> 113 ccggtgccgc caccatcccc tgacccacgc ccctgacccc tcccaaggag acgaccttcc 60 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta 120 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac 180 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 240 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 300 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 360 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 420 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 480 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 540 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 600 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 660 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac 720 cgaagccgac ccgggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta 780 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 840 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat 900 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 960 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 1020 gatccgcgcc ggatccttaa ttaagtctag agtcgactgt ttaaacctgc aggcatgcaa 1080 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 1140 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 1200 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 1260 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 1320 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 1380 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 1440 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 1500 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 1560 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1620 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 1680 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 1740 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 1800 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 1860 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1920 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 1980 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 2040 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 2100 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 2160 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 2220 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 2280 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 2340 S

agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 2400 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 2460 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 2520 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 2580 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 2640 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 2700 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 2760 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 2820 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 2880 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 2940 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3000 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3060 gaaggcaaaa tcccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 3120 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 3180 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 3240 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 3300 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 3360 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 3420 agggcgcggc agcgggtgtt ggcgagtgtc ggggctggct taactatgcg gcatcagagc 3480 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 3540 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 3600 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 3660 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcg 3720 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3780 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3840 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3900 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3960 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 4020 agtagtgagg aggctttttt ggaggctcgg tacccccttg cgctaatgct ctgttacagg 4080 tcactaatac catctaagta gttgattcat agtgactgca tatgttgtgt tttacagtat 4140 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac 4200 gtttctcgtt cagctttttt atactaagtt ggcattataa aaaagcattg cttatcaatt 4260 tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgatt tcaattttgt 4320 cccactccct gcctctgggg ggcgcg 4346 <210> 114 <211> 3166 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeo Plasmid <400> 114 cagttgccgg cCgggtcgcg caggccgaac tcccggcccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc cttcagagaa gcttgcatgc ctgcaggtcg 420 actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgcgaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctctttc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tggggcgagc ggtatccgct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga caagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt caaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740 ggccccagtg ctgcaacgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccaatcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcatccagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tagttaacaa aaaaaagccc 2640 gccgaagcgg gctttattac caagcgaagc gccattcgcc attcaggctg cgcaactgtt 2700 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 2760 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 2820 cggccagtcc gtaatacgac tcacttaagg ccttgactag agggtcgacg gtatacagac 2880 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 2940 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 3000 caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc 3060 ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa 3120 atctcgtagc acgtgtcagt cctgctcctc ggccacgaag tgcacg 3166 <210> 115 <211> 7600 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeo3'6XHS4eGFP Plasmid <400> 115 cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtccccgaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatcta gttattaata 420 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 480 tacggtaaat ggcccgagtg gctgaccgcc caacgacccc cgcccattga cgtcaataat 540 gacgtatgtt cccatagtaa ccccaatatg gactttccat tgacgtcaat gggtggacta 600 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 660 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 720 ggactttcct acttggcagt acatctacgt attaatcatc gctattacca tgggtcgagg 780 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 840 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg 900 ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca 960 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 1020 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc 1080 ccggttcgcg ccacctcacg ccccccaccc cggctctgac tgaccgcgttactcccacag 1140 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 1200 ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc 1260 gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg 1320 cccgcgctgc ccgccggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 1380 gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg 1440 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc 1500 ggtcgggctg taacgccccc cttcaccccc ctccccgagt tgctgagcac ggcccggctt 1560 cgggtgcggg gctccgtgcg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc 1620 ggcaggtggg ggtgccgggc ggggcggggc cgcatcggcc cggggagggc tcgggggagg 1680 ggcgcggcgg ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct 1740 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct ggcggagccg 1800 aaatctggga ggcgccgccg caccccctct agcggacgcg ggcgaagcgg tgcggcgccg 1860 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc 1920 atctccagcc tcggggctgc cgcaggggga cggctgcctt cggggaggac ggggcagggc 1980 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 2040 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt 2100 tggcaaagaa ttcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 2160 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 2220 cgagcgcgat gccacctacg gcaagctgac cctgaagttc atctccacca ccggcaagct 2280 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg 2340 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccattcccg aaggctacgt 2400 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 2460 gttcgaaggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 2520 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 2580 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 2640 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 2700 gctgctgccc gacaaccact acctgaccac ccagtccgcc ctgagcaaag accccaacga 2760 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 2820 ggacgagctg tacaagtaag aattcactcc tcaggtgcag gctgcctatc agaaggtggt 2880 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa 2940 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt 3000 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg 3060 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc 3120 catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc 3180 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta 3240 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac 3300 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat 3360 gaagatccct cgacctgcag cccaagcttg catgcctgca ggtcgactct agtggatccc 3420 ccgccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 3480 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgct:gcc ggctcgggga 3540 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 3600 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 3660 cggagccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 3720 aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccggacgctg ccggctcggg 3780 gatgcgcggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 3840 gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 3900 tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 3960 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcaggc tgccgcgtcg 4020 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 4080 gcgggggagg gacgtaatta catccctggg ggctctgggg gggggctgtc cccctgagcg 4140 gatccgcggc cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca 4200 gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct 4260 cggggatggg gggggagggc cgaaccggag cggagccccg ggcggctcgc tgctgccccc 4320 tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag 4380 cggaaccgcg gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt 4440 cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg 4500 ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc 4560 cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg 4620 agcgaatccg cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg 4680 ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc 4740 ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc 4800 cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg 4860 tgagcggatc cgcggggctg caggaattcg taatcatggt catagctgtt tcctgtgtga 4920 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 4980 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5040 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5100 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5160 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatga 5220 gaggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5280 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5340 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5400 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5460 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5520 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5580 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5640 = s ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5700 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 5760 gctctgctga agCcagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5820 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5880 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 5940 tcacgttaag ggattttggt catgagatta tcaaaaagca tcttcaccta gatcctttta 6000 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6060 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6120 gttgcctgac tccccgtcgt gtagataact acgatacgcg agggcttacc atctggcccc 6180 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6240 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6300 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6360 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6420 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6480 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6540 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6600 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6660 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6720 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6780 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6840 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6900 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6960 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7020 ccgcgcacat ttccccgaaa agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa 7080 gcgggcttta ttaccaagcg aagcgccatt cgccattcag gctgcgcaac tgttgggaag 7140 ggcgatcggt gcgggcttct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 7200 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 7260 gtccgtaata cgactcactt aaggccttga ctagagggtc gacggtatac agacatgata 7320 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 7380 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 7440 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc 7500 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg 7560 tagcacgtgt cagtcctgct cctcggccac gaagtgcacg 7600 <210> 116 <211> 7631 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeos'6XHS4eGFP Plasmid <400> 116 cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420 agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccattccg gtccggcgct 540 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660 acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900 atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020 cgctgccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140 ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gcccgggtcc 1260 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380 ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcctgg gcacggggaa 1560 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620 tgggggatac ggggccgcgg atccgctcac ggggacagcc ccccccgaaa gcccccaggg 1680 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740 gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800 aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860 cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccggctg 1980 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt Cccatagtaa 2040 cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100 tgccagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220 acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340 ttttgtgcag cgatgggggc gggggcgggg ggggcgcgcg ccacccgggg cggggcgggg 2400 cgaggggcgg ggcggggcga ggcggagagg tgcggggcca gccaatcaga gcggcgcgct 2460 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520 gcggcggggg ggaatcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 25B0 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700 gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880 gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060 gggcgtggcg cggggctcgc cgtgccgggc gggggggggc ggcaggtggg ggtgccgggc 3120 ggggcggggc cgtctcgggc cggggagggc tcgggggagg gcggcggcgg ccccggagcg 3180 ccggcggctg tcgaggcgcg gcgagccgga gccattgcct tttatggtaa tcgtgcgaga 3240 gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300 caccccctat agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360 gagggccttc gtgcgtcgcc gcggcgcggt ccccttctcc atctccagcc tcggggctgc 3420 cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480 tgaccgccgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540 tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660 gcgacgtaaa cggccacaag ttcagcgtgt ccggccaggg cgagggcgat gccacctacg 3720 gcaagctgac cctaaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140 accactacca gcagaacacc cccatcggcg acggccccgt gctgccgccc gacaaccact 4200 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320 aattcactcc tcaggtgcag gctgcctatc aaaaggtggt ggctggtgtg gccaatgccc 4380 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560 atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620 caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860 cccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc 4920 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4980 catacgagcc ggaagcataa agtgtaaagc ctgggctgcc taatgagtga gctaactcac 5040 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 5100 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 5160 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5220 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5280 aaaaggccag caaaaggcca ggaaccgtaa aaaagccgcg ttgctggcgt ttttccatag 5340 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5400 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5460 i =.

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5520 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5580 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5640 tgagtccaac ccggtaaaac acgacttatc gccactggca gcagccactg gtaacaggat 5700 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5760 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5820 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5880 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5940 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 6000 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 6060 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 6120 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 6180 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6240 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6300 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6360 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6420 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6480 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6540 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6600 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6660 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6720 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6780 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6840 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6900 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6960 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 7020 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 7080 tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt attaccaagc gaagcgccat 7140 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 7200 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 7260 tcccagtcac gacgttgtaa aacgacggcc agtccgtaat acgactcact taaggccttg 7320 actagagggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac 7380 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 7440 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat 7500 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt 7560 catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca 7620 cgaagtgcac g 7631 <210> 117 <211> 4615 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeo6XHS4 Plasmid <400> 117 cagttgccgg ccaggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccggaagaa cccgagccgg tcggtccaga actcgaccgc tccggccacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgcatgc ctgcaggtcg 420 actctagtgg accccccgcc ccgtatcccc caggtgtctg caggctcaaa gagcagcgag 480 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg 540 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct 600 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt 660 ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 720 agaagcgttc agaggaaagc gatcccgtgc cagtttcccc gtgcccgggc tgtccccgca 780 cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 840 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg gggggggggt 900 gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 960 cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 1020 cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 1080 cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 1140 ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 1200 S

agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 1260 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggggg 1320 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 1380 ggctgtcccc gtgagcggat ccccgtcccc gtatccccca ggtgtctgca ggctgaaaga 1440 gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc'ttccccgtgc ccgggctgtc 1500 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg acctgagcgg agccccgggc 1560 ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 1620 ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc cacgtttctg caggctcaaa 1680 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 1740 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 1800 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 1860 ggggggctgt ccccgtgagc ggatccgcgg ggctgcagga attcgtaatc atggtcatag 1920 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1980 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2040 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2100 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 2160 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2220 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2280 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2340 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2400 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2460 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2520 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2580 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2640 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2700 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2760 gtatttggta tctgcgctct gctgaaccca gttaccttcg gaaaaagagt tggtagctct 2820 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2880 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2940 cagtggaacg aaaattgacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3000 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tttaaagtat atatgagtaa 3060 acttggtctg acaattacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3120 tttcgttcat ccatagttgc =ctgactcccc gtcgtgtaga taactacgat acgggagggc 3180 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3240 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3300 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3360 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3420 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3480 ttgtgcaaaa aagcggttag ctcgttcggt cctccgatcg ttgtcagaag taagttggcc 3540 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3600 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3660 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3720 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3780 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3840 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3900 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3960 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4020 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt agttaacaaa 4080 aaaaagcccg ccgaagcggg ctttattacc aagcgaagcg ccattcgcca ttcaggctgc 4140 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 4200 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 4260 gtaaaacgac ggccagtccg taatacgact cacttaaggc cttgactaga gggtcgacgg 4320 tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 4380 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 4440 tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat 4500 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt 4560 ggaatcgaaa tctcgtagca cgtgtcagtc ctgctcctcg gccacgaagt gcacg 4615 <210> 118 <211> 17384 <212> DNA
<213> Artificial Sequence <220>
<223> pFK161 Plasmid <400> 118 . = . .

gcgcacgagg gaccttccag ggggaaacgc ctggtatctt tatagtcctg tcggggtttc 60 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 120 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 180 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 240 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 300 aagagcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat 360 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc 420 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac 480 aggagcacga tcatgcacac ccgtcagatc cagacatgat aagatacatt gatgagtttg 540 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 600 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 660 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 720 acaaatgtgg tatggctgat tatgatctct agtcaaggca ctatacatca aatattcctt 780 attaacccct ttacaaatta aaaagctaaa ggtacacaat ttttgagcat agttattaat 840 agcagacact ctatgcctgt gtggagtaag aaaaaacagt atgttatgat tataactgtt 900 atgcctactt ataaaggtta cagaatattt ttccataatt ttcttgtata gcagtgcagc 960 tttttccttt gtggtgtaaa tagcaaagca agcaagagtt ctattactaa acacagcatg 1020 actcaaaaaa cttagcaatt ctgaaggaaa gtccttgggg tcttctacct ttctcttctt 1080 ttttggagga gtagaatgtt gagagtcagc agtagcctca tcatcactag atggcatttc 1140 ttctgagcaa aacaggtttt cctcattaaa ggcattccac cactgctccc attcatcagt 1200 tccataggtt ggaatctaaa atacacaaac aattagaatc agtagtttaa cacattatac 1260 acttaaaaat tttatattta ccttagagct ttaaatctct gtaggtagtt tgtccaatta 1320 tgtcacacca cagaagtaag gttccttcac aaagatccgg accaaagcgg ccatcgtgcc 1380 tccccactcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat 1440 gccgacggat ttgcactgcc ggtagaactc gcgaggtcgt ccagcctcag gcagcagctg 1500 aaccaactcg cgaggggatc gagcccgggg tgggcgaaga actccagcat gagatccccg 1560 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 1620 aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt 1680 tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct 1740 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa 1800 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca 1860 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc 1920 aggcatcgcc atcggtcacg acgagatcct cgccgtcggg atgcgcgcct tgagcctggc 1980 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacagg 2040 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2100 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2160 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2220 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2280 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc 2340 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2400 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2460 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2520 atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2580 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2640 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactacaag ctacctgctt 2700 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2760 tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg 2820 ccctgagtgc ttgcggcagc gtgaaagctt tttgcaaaag cctaggcctc caaaaaagcc 2880 tcctcactac ttctg aata gctcagaggc cgaggcggcc taaataaaaa aaattagtca 2940 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag 3000 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg 3060 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact 3120 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagccg 3180 gatctgcagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac 3240 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 3300 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg 3360 tcgaggtggc ccggctccat gccccgcgac gcaacgcggg gaggcagaca aggtataggg 3420 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcgcat aaatcgccgt 3480 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag 3540 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctccaacg cggcatcccg 3600 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac 3660 gccagcaaga cgtagcccag cgcgtcgggc cgccatgccg gcgataatgg cctgcttctc 3720 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 3780 gaataccgca agcgacaggc cgatcatcgt cccgcttcag cgaaagcggt cctcgccgaa 3840 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 3900 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 3960 tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4020 =

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4080 gcccaacagt cccccggcca cggtgctgcc actataccca cgccgaaaca agcgctcatg 4140 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca 4200 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcttggca 4260 gtcacagcat gcgcatatcc atgcttcgac catgcgctca caaagtaggt gaatgcgcaa 4320 tgtagtaccc acatcgtcat cgctttccac tgctctcgcg aataaagatg gaaaatcaat 4380 ctcatggtaa tagtccatga aaatccttgt attcataaat cctccaggta gctatatgca 4440 aattgaaaca aaagagatgg tgatctttct aagagatgat ggaatctccc ttcagtatcc 4500 cgatggtcaa tgcgctggat atgggataga tgggaatatg ctgattttta tgggacagag 4560 ttgcgaactg ttcccaacta aaatcatttt gcacgatcag cgcactacga actttaccca 4620 caaatagtca ggtaatgaat cctgatataa agacaggttg ataaatcagt cttctacgcg 4680 catcgcacgc gcacaccgta gaaagtcttt cagttgtgag cctgggcaaa ccgttaactt 4740 tcggcggctt tgctgtgcga caggctcacg tctaaaagga aataaatcat gggtcataaa 4800 attatcacgt tgtccggcgc ggcgacggat gttctgtatg cgctgttttt ccgtggcgcg 4860 ttgctgtctg gtgatctgcc ttctaaatct ggcacagccg aattgcgcga gcttggtttt 4920 gctgaaacca gacacacagc aactgaatac cagaaagaaa atcactttac ctttctgaca 4980 tcagaagggc agaaatttgc cgttgaacac ctggtcaata cgcgttttgg tgagcagcaa 5040 tattgcgctt cgatgacgct tggcgttgag attgatacct ctgctgcaca aaaggcaatc 5100 gacgagctgg accagcgcat tcgtgacacc gtctctttcg aacttattcg caatggagtg 5160 tcattcatca aggacgccgc tatcgcaaat ggtgctatcc acgcagcggc aatcgaaaca 5220 cctcagccgg tgaccaatat ctacaacatc agccttggta tccagcgtga tgagccagcg 5280 cagaacaagg taaccgtcag tgccgataag ttcaaagtta aacctggtgt tgataccaac 5340 attgaaacgt tgatcgaaaa cgcgctgaaa aacgctgctg aatgtgcggc gctggatgtc 5400 acaaagcaaa tggcagcaga caagaaagcg atggatgaac tggcttccta tgtccgcacg 5460 gccatcatga tggaatgttt ccccggtggt gttatctggc agcagtgccg tcgatagtat 5520 gcaattgata attattatca tttgcgggtc ctttccggcg atccgccttg ttacggggcg 5580 gcaacctcgc gggttttcgc tatttatgaa aattttccgg tttaaggcgt ttccgttctt 5640 cttcgtcata acttaatgtt tttatttaaa ataccctctg aaaagaaagg aaacgacagg 5700 tgctgaaagc gagctttttg gcctctgtcg tttcctttct ctgtttttgt ccgtggaatg 5760 aacaatggaa gtcaacaaaa agcagctggc tgacattttc ggtgcgagta tccgtaccat 5820 tcagaactgg caggaacagg gaatgcccgt tctgcgaggc ggtggcaagg gtaatgaggt 5880 gctttatgcc tctgccgccg tcataaaatg gtatgcggaa agggatgctg aaattgagaa 5940 cgaaaagctg cgccgggagg ttgaagaact gcggcaggcc agcgaggcag atccacagga 6000 cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga 6060 ctgggcggcg gcaaagcggt cggacagtgc tccgagaacg ggtgcgcata gaaattgcat 6120 caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg aatggacgat 6180 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt 6240 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg 6300 ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gcggtcaaac 6360 atgagaattc gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420 cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc ctgcccgacg ctgccccgcg 6480 cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540 tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600 ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660 gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720 gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780 tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840 tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900 ggtggcggat ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960 gagaggcctg gctttcgggg ggcaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020 aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080 ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140 gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200 ggaaatggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260 ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320 ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380 ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440 gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggctggggtg 7500 tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560 tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620 ctgggccgcg cgggttgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680 ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740 cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800 gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860 gccgctcggg ggtcttcgtc ggtacgcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920 cggtgtcgcc tcctagggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980 ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040 = =, tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100 gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160 ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220 acccgtggcc gtgctgtggg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280 cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcggaag 8340 gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400 gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460 agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520 ccggcccttt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580 accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640 gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcgtccttcc 8700 cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760 catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820 tcgccggggg ctggccgttg tccggtcttt cctgcccgac ccccgttgcc gtggtcttct 8880 ctcgccggct tcgcggactc ctggctttgc ccggagggtc agggggcttc ccggttcccc 8940 gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000 gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtgagttgg gccctggcgt 9060 tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120 gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180 tcgttggtgt ggggagtgaa tggtgctacc ggtcatttcc tcccgcgtgg tttgactgtc 9240 tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300 cggcccggtg cggtcgacgt tccggctttc ccgatgccga ggggttcggg atttgtgccg 9360 gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420 gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480 tccggccgca tgcactctcc cgttccgcgc gagcgcctgc ccggctcacc cccggtttgt 9540 cctcccgcga ggctctgctc cgccgccgcc tcctcctcct ctctcgcgct ctctctcccg 9600 cctggtcctg tcccaccccc gacgttccgc tcgccgttcc ttacctggtt gatcctgcca 9660 ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720 agtgaaactg ccaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780 ctacttggat aactgtggta attctagagc taatacat-gc cgacgggcgc tgacccccct 9840 tcccgggtgg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900 ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960 acgccccccg tggcggcgaC gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020 tcgccgtgcc taccatggtg accacggg g acggggaatc agggttcgat tccggagagg 10080 gagcctgaga aacggctaCC acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140 cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200 ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260 cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320 tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380 ccccttgcct ctcggcgtcc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440 gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500 aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560 taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620 gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca aaaacgaaag 10680 tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740 cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800 ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860 ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920 cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980 ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040 ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100 gcgtccagcc acccgagatt gagcaataac aggtctgtga tgccettaga tgtccggggc 11160 tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220 aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280 gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340 accgcccgtc gctactaccg attggatggt ttagtgaggc cctctgaccg gccccgccgg 11400 ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460 agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520 ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgcgagg 11580 cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640 gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700 tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760 gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820 ggtttttgac cccgtccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880 cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940 ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000 gtcctccccg ctcctgtccc gggtacctag Ctgtcgcgtt ccggcgcgga ggtttaagga 12060 = =

ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120 cggtcgttcg ggcgcctctc cctcagactc catgaccctc ctcccccccc tgccgccgtt 12180 ccccgggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctccggc gccgtggggg 12240 cccgaccccc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300 gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360 cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420 cctctctctc cggggaaagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480 cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540 tgcgtcgatg aataacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600 catcgacact tcgaacgcac ttgcggcccc gggttcctcc Cggggctacg cctgtctgag 12660 cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720 ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780 gggcggttgt cggtgttgcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840 cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900 gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960 tgggaaccca ccgcgccccc gtggcgcccg ggggtgtgcg cgtccgcatc tgctctggtc 13020 gaggttggcg gttgagggtg ttcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080 ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140 gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200 ggcgccccgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260 gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320 cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380 cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440 ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500 gggagggaga gggcctCgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560 ctgtgggctg tgcgtcccgg gggttgCgtg tgagtaagat cctccacccc cgccgccctc 13620 ccctcccgcc ggcctctcgg ggaccccctg agacagttcg ccggctcgtc ctcccgtgcc 13680 gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740 tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800 attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860 gaagagccca gcgccgaatc cccgcccctc gtcccgccgt gggaaatgtg gcattcggaa 13920 gacccactcc ccggcgccgc tcgttggggg cccaagtcct tctgatcgag gcccagcccg 13980 tggacggtgt gaggccggta gcggccccgg cgcgccgggc ttcggtcttc ccggagtcgg 14040 gttgcttggg aatgcagccc aaagctgttg gtaaactcca tctaaggcta aataccggca 14100 cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160 tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220 gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280 ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340 gcctccggcg gcgggcgcgg ggggtggtgt ggtggtgccg cgcgggcggg-gccgggggtg 14400 gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460 ggcggtgcgc cgcgaccggc tccgggacgg cctggaatgc ccggtgggga aggtggctcg 14520 gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580 ggccgcgctt tcgccgaatc cctgggccga ggaagccaga tacccgtcgc cgcgctctcc 14640 ctctcccccc gtccgcctcc cgggcggtcg tgggggtggg gtccgggcgg cccctcccac 14700 ggcgagaacg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760 cgcactctcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccgtg gggaccgtcg 14820 gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatatcg gctacccacc 14880 cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940 gaaaggcccc gtggcgcaat gaaggtgaag ggccccgccc ggcgccccga ggtgggatcc 15000 cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060 aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120 cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180 cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240 tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300 aatgattaga ggtcttgggg ccaaaacgat ctcaacctat tctcaaactt taaatgggta 15360 agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420 ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480 gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540 gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600 aatggatggc gctggagcgt cgggcccata cccgcccgtc gccgcagtcg gaacggaacg 15660 ggacgggagc ggccgcgaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat 15720 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 15780 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 15840 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 15900 atttccgtgt cgcccttatt cccttttttg cggcattttg cttcctgttt ttgctcaccc 15960 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 16020 cgaactggat Ctcaacagcg gtaagatcct tgagagtttt Cgccccgaag aacgttttcc 16080 = = , aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 16140 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 16200 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 16260 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 16320 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 16380 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc 16440 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 16500 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 16560 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 16620 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 16680 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 16740 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 16800 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 16860 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 16920 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 16980 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 17040 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 17100 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 17160 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 17220 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 17280 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc cgaagggaga 17340 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gaga 17384 <210> 119 <211> 2814 <212> DNA
<213> Artificial Sequence <220>
<223> pLITMUS38 Plasmid <400> 119 gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300 gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420 acactattct cagaatgact tggttgagta ctcaCCagtc acagaaaagc atcttacgga 480 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaatat 600 gggggatcat gttactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020 ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080 aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200 aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac caaactaaga 2040 tacctacagc gtgagctatg agaaagcgcc acccttcccg aagggagaaa ggcggacagg 2100 =

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280 ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460 ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gatatctgga 2520 tccacgaatt cgctagcttc ggccgtgacg cgtctccgga tgtacaggca tgcgtcgacc 2580 ctctagtcaa ggccttaagt gagtcgtatt acggactggc cgtcgtttta caacgtcgtg 2640 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 2700 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2760 atggcgaatg gcgcttcgct tggtaataaa gcccgcttcg gcgggctttt tttt 2814 <210> 120 <211> 2847 <212> DNA
<213> Artificial Sequence <220>
<223> pLIT38attB Plasmid <400> 120 gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300 gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagtgct 360 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780 aattgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840 tgaagctggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020 ctcatatata ctttagattg atttaccccg gttgataatc agaaaacccc caaaaacagg 1080 aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200 aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacacggc gcgtaaaagg 1560 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tcaattttcg 1620 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740 ccggatcaag agctaccaac tttttttcgg aaggtaactg gcttcagcag agcgcagata 1800 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280 ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460 ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520 tgctttttta tactaacttg agcgaaatct ggatccacga attcgctagc ttcggccgtg 2580 acgcgtctcc ggatgtacag gcatgcgtcg accctctagt caaggcctta agtgagtcgt 2640 attacggact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 2700 = =

ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 2760 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttc gcttggtaat 2820 aaagcccgct tcggcgggct ttttttt 2847 <210> 121 <211> 4223 <212> DNA
<213> Artificial Sequence <220>
<223> pLIT38attBBSRpolyA2 Plasmid <400> 121 accatgaaaa catttaacat ttctcaacaa gatctagaat tagtagaagt agcgacagag 60 aagattacaa tgctttatga ggataataaa catcatgtgg gagcggcaat tcgtacgaaa 120 acaggagaaa tcatttcggc agtacatatt gaagcgtata taggacgagt aactgtttgt 180 gcagaagcca ttgcgattgg tagtgcagtt tcgaatggac aaaaggattt tgacacgatt 240 gtagctgtta gacaccctta ttctgacgaa gtagatagaa gtattcgagt ggtaagtcct 300 tgtggtatgt gtagggagtt gatttcaaac tatgcaccag attgttttgt gttaatagaa 360 atgaatggca agttagtcaa aactacgatt gaagaactca ttccactcaa atatacccga 420 aattaaaagt tttaccatac caagcttggc tgctgcctga ggctgaacga cctcgcggag 480 ttctaccggc agtgcaaatc cgtcggcatc caggaaacca gcagcggcta tccgcgcatc 540 catgcccccg aactgcagga gtggggaggc acgatggccg ctttggtccg gatctttgtg 600 aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc 660 tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt 720 tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg gaatgccttt 780 aatgaggaaa acctgttttg ctcagaagaa atgccatcta gtgatgatga ggctactgct 840 gactctcaac attctactcc tccaaaaaag aagagaaagg tagaagaccc caaggacttt 900 ccttcagaat tgctaagttt tttgagtcat gctgtgttta gtaatagaac tcttgcttgc 960 tttgttattt acaccacaaa ggaaaaagct gcactgctat acaagaaaat tatggaaaaa 1020 tattctgtaa cctttataag taggcataac agttataatc ataacatact gttttttctt 1080 actccacaca ggcatagagt gtctgctatt aataactatg ctcaaaaatt gtgtaccttt 1140 agctttttaa tttgtaaagg ggttaataag gaatatttga tgtatagtgc cttgactaga 1200 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca 1260 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 1320 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagatccaga 1380 tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg cagagaagct tggcgccagc 1440 cggcttcaat tgcacgggcc ccactagtga gtcgtattac gtagcttggc gtaatcatgg 1500 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1560 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attacatgtg 1620 agcaaaaggc cagcaaaagg caaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1680 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1740 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tacgctctcc 1800 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1860 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1920 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttattcg gtaactatcg 1980 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagaagcca ctggtaacag 2040 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2100 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2160 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2220 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2280 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2340 attatcaaaa aggatcttca cctagatcct tttacgcgcc ctgtagcggc gcattaagcg 2400 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2460 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc tttccccgtc aagctctaaa 2520 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2580 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2640 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2700 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 2760 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 2820 aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 2880 ggtaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 2940 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3000 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 3060 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3120 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3180 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct'3240 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3300 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3360 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 3420 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 3480 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 3540 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggagaacgt 3600 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 3660 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 3720 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 3780 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 3840 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3900 cgaaaagtgc cacctgacgt agttaacaaa aaaaagcccg ccgaagcggg ctttattacc 3960 aagcgaagcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 4020 cctcttcgct attacgccag ctagcgaaag ggggatgtgc tgcaaggcga ttaagttggg 4080 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtccg taatacgact 4140 cacttaaggc cttaactata gggtcgacgc atgcctgtac atccggagac gcgtcacggc 4200 cgaagctagc gaattagtgg atc 4223 <210> 122 <211> 2686 <212> DNA
<213> Artificial Sequence <220>
<223> pUC1B Plasmid <400> 122 tcgcgcgttt cggtgattac ggtgaaaacc tctgacacat gaagctcccg gaaacggtca 60 cagcttgtct gttaggggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggaaaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420 actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaat.acg gttatccaca 780 gaatcagggg ataacgcagg aaacaaaatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccatg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgctttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggtggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattataaa aaaggat:ctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga taggggaggg cttaccatct 1740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccagtcta ttaattgttg Ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg cctcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggagttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686 <210> 123 <211> 8521 <212> DNA
<213> Artificial Sequence <220>
<223> pCXeGFPattB(6xHS4)2 Plasmid <400> 123 tacggggcgg gggatccact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttatggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaagg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg gggacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgtcggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acgagtgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1920 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2040 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280 agcagaacac ccccatcggc gacgcccccg tgctgctgcc cgacaaccac tacctgagca 2340 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 2700 agtatttgtt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 2940 ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120 = =

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360 ggaggctagt ggatcccccg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 3420 agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 3480 cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 3540 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct 3600 gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 3660 cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 3720 cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcgctt 3780 cgctgctgcc ccctggcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 3840 ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 3900 agcgagaagc gttcagagga aagcgattcc gtgccacctt ccccgtaccc gggctgtccc 3960 cgcacgctgc cgattcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 4020 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 4080 ggctgtcccc gtgagcggat ccgcggcccc gtatccccca ggtgtctgca ggctcaaaga 4140 gcagcgacaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc 4200 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc 4260 ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 4320 ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc caggtgtctg caggctcaaa 4380 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 4440 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 4500 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 4560 ggggggctgt ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca 4620 aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc 4680 tgtccccgca cgctgccggc.tcggggatgc ggggggagcg ccggaccgga gcggagcccc 4740 gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg 4800 ggggggggct gtccccgtga gcggatccgc ggggctgcag gaattcgatt gaagcctgct 4860 tttttatact aacttgagcg aaatcaagct cctaggcttt tgcaaaaagc taacttgttt 4920 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4980 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5040 tggatccgct gcattaatga atcggccaac gcgcggggag aggcgctttg cgtattgggc 5100 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5160 tatcagctca ctcaaaggcg gtaataccgt tatccacaga atcaggggat aacgcaggaa 5220 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5280 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5340 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5400 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtcCgcCttt ctcccttcgg 5460 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 5520 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5580 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5640 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5700 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5760 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5820 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5880 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5940 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6000 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 6060 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 6120 tcgtgtagat aactacgata cggga ggct taccatctgg ccccagtgct gcaatgatac 6180 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 6240 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 6300 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 6360 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6420 gataaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 6480 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6540 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 6600 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6660 tacgggataa taccgtgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6720 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6780 ctcgtgcacc caactgatct tcagaagctt ttactttcac cagcgtttct gggtgagcaa 6840 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6900 tcatactctt cctttttcaa tattattgaa gcatttatga gggttattgt ctcatgggcg 6960 gatacatatt tgaatgtatt tagaaaaata aaCaaatagg ggttccgcgc acatttcccc 7020 gaaaagtgcc acctggtcga cggtatcgat aagcttgata tcgaattcct gcagccccgc 7080 ggatccgctc acagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc 7140 = , cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc 7200 atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc 7260 tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg atacggggcc 7320 gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct 7380 cccccgctag ggggcagcag cgagccgccc ggggctccgc=tccggtccgg cgctcccccc 7440 gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc 7500 gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg ggatacgggg 7560 ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc 7620 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 7680 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 7740 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg ggggatacgg 7800 ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt 7860 ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc 7920 ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg 7980 gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tgggggatac 8040 ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 8100 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 8160 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 8220 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 8280 acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 8340 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 8400 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 8460 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 8520 a 8521 <210> 124 <211> 8851 <212> DNA
<213> Artificial Sequence <220>
<223> p18EPOcDNA Plasmid <400> 124 cagttgccgg ccgggtcgcg cagggcgaac tccccccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420 agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660 acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900 atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140 ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380 ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620 tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740 gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1600 aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860 cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040 cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220 acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340 ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg cgaggcggcg cgggtcgggg 2400 cgaggggcgg gccgcggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520 gcggcgggcg ggagtCgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580 ccccccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640 ttctactccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700 gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760 gtgcgtgcgt gtgtgtgtgc ggggggagcg ccgcgtggcg cccgcgctgc ccggcggctg 2820 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880 gccgg ggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000 ctgcaccccc ctccccgagt tgctgagcac ggcccagctt cgggtgcggg gctccgtgcg 3060 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg gttgccgggc 3120 gggggggggc cgcctcgggc cggggagggc tcggtcgagg ggcgcggcgg ccccggagcg 3180 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa ttgtgcgaga 3240 gggcgcagcg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300 caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420 cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480 tgaccggcgg ctctagaatg ggggtgcacg aatgtcctgc ctggctgtgg cttctcctgt 3540 ccctgctgtc gctccctctg ggcctcccag tcctgggcgc cccaccacgc ctcatctgtg 3600 acagccgagt cctggagagg tacctcttgg agctcaagga ggccgagaat atcacgacgg 3660 gctgtgctga acactgcagc ttgaatgaga atatcactgt cccagacacc aaagttaatt 3720 tctatgcctg gaagaggatg gaggtcgggc agcaggccgt agaagtctgg cagggcctgg 3780 ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt ggtcaactct tcccagccgt 3840 gggagcccct gcagctgcat gtggataaag ccgtcagtgg ccttcgcagc ctcaccactc 3900 tgcttcgggc tctgggagcc cagaaggaag ccatctcccc tccagatgcg gcctcagctg 3960 ctccactccg aacaatcact gctgacactt tccgcaaact cttccgagtc tactccaatt 4020 tcctccgggg aaagctgaag ctgtacacag gggaggcctg caggacaggg gacagatgac 4080 gtacaagtaa gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt 4140 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg 4200 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat 4260 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa 4320 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg ccatatgctg 4380 gctgccatga acaaaggtgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 4440 tc:attcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 4500 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 4560 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tgaagatccc 4620 tcgacctgca gcccaagctt gcatgcctgc aggtcgactc tagtggatcc cccgccccgt 4680 atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc 4740 gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg 4800 agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac 4860 gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat ccgcggcccc 4920 gtatccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc 4980 ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg 5040 ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cggggaaggg 5100 acgtaattac atccctgggg gctttggggg ggggctgtcc ccgtgagcgg atccgcggcc 5160 ccgtatcccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga 5220 tcccgtgcca ccttccccgt ggtcgggctg tccccgcacg ctgccggctc ggggatgcgg 5280 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag 5340 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtgagc ggatccgcgg 5400 ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc 5460 gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc 5520 ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcgggtg 5580 agggacgtaa ttacatccct gggggctttg ggggggggct gtccccgtga gctgatcccc 5640 ggccccgtat cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa 5700 gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat 5760 gcggggggag cgccggaccg gagcggatcc ccgggcggct cgctgctgcc ccctggcggg 5820 ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccccgt gagcggatcc 5880 gcggccccgt atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga 5940 aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg 6000 =

atgcgggggg agcgccggac cggagcggag Ccccgggcgg ctcgctgccg ccccctagcg 6060 ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat 6120 ccccggggct gcaggaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6180 ccgctcacaa ttccacacaa catatgaccc ggaagcataa agtgtaaagc ctggggtgcc 6240 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6300 aacctgtcgt gccagctgca taaatgaacc ggccaacgcg cggggagagg cggtttgcgt 6360 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6420 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6480 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6540 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6600 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6660 tccctcgtgc gctctcctgt tccgaccctg.ccgcttaccg gatacctgtc cgcctttctc 6720 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6780 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6840 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6900 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6960 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 7020 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 7080 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7140 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7200 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7260 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 7320 ttaatcagtg aggaacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 7380 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 7440 atgataacgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 7500 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7560 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7620 attgctacag gcatCgtggt gtcacgctcg tcgtttggta ttgctttatt cagctccggt 7680 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7740 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7800 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7860 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgtccg 7920 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7980 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tcttgagatc cagttcgatg 8040 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 8100 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 8160 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 8220 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 8280 tttccccgaa aagtgccacc tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt 8340 attaccaagc gaagcgccat tcgccattca gtttgcgcaa ctgttgggaa gggcgatcgg 8400 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 8460 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtccgtaat 8520 acgactcact taaggccttg actagagggt cgacggtata cagacatgat aagatacatt 8580 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 8640 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc 8700 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac 8760 gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg 8820 tcagtcctgc tcctcggcca cgaagtgcac g 8851 <210> 125 <211> 10474 <212> DNA
<213> Artificial Sequence <220>
<223> pl8genEPO Plasmid <400> 125 cagttgccgg ccaggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgttccaga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420 agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660 acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720 acgtccctcc cccgctaggg ggcagcagcg aaccgcccgg ggctccgctc cggtccggcg 780 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900 atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140 ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260 ggcgctcccc ccgcatgccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380 ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggat ccgctcacgt 1500 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620 tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaca gcccccaggg 1680 atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740 gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800 aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860 cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920 tagttcatag cccatataag gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040 cgccaatagg gactttccat tgccgtcaat gggtggacta tttacggtaa actgcccact 2100 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220 acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340 ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400 cgaggggcgg ggcggggcga ggcgaagatg tgcggcggca gccaatcaga gcggcgcgct 2460 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520 gcagcgtgcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700 gcgtgaaagc cttaaagggc tccgggacgg ccctttgtgc ggggggtagc ggctcggggg 2760 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880 gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120 ggggcgtggc cgcctcgggc cgggcagggc tcgggggagg ggcgcggcgg ccccggagcg 3180 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240 gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300 caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420 cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480 tgaccggcgg ctctagatgc atcctcgagc ggccgccagt gtgatggata tctgcagaat 3540 tcgccctttc tagaatgggg gtgcacggtg agtactcgcg ggctgggctc tcccgcccgc 3600 ccgggtccct gtttgagcgg ggatttagcg ccccggctat tggccaggag gtggctgggt 3660 tcaaggaccg gcgacttgtc aaggaccccg gaagggggag gggggtgggg tgcctccacg 3720 tgccagcggg gacttggggg agtccttggg gatggcaaaa acctgacctg tgaaggggac 3780 acagtttggg ggttgagggg aagaaggttt gggggttctg ctgtgccagt ggagaggaag 3840 ctgataagct gataacctgg gcgttaaagc caccacttat ctgcca.gagg ggaagcctct 3900 gtcacaccag gattgaagtt tggccggaga agtggatgct gttagctggg ggtggggtgt 3960 gcacacggca gcaggattga atgaaggcca gggaggcagc acctgagtgc ttgcatggtt 4020 ggggacagga aggacgagct ggggcagaga cgtggggatg aaggaagctg tccttccaca 4080 gccacccttc tccctccccg cctgactctc agcctggcta tctgttctag aatgtcctgc 4140 ctggctgtgg cttctcctgt ccctgctgtc gctccctctg ggcctcccag tcctgggcgc 4200 cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 4260 ggccgagaat atcacggtga gaccccttcc ccagcacatt ccacagaact cacgctcagg 4320 gcttcaggga actcctccca gatccaggaa cctggcactt ggtttggggt ggagttggga 4380 agctagacac tgccccccta cataagaata agtctggtgg ccccaaacca tacctggaaa 4440 ctaggcaagg agcaaagcca gcagatccta cggcctgtgg gccagggcca gagccttcag 4500 ggacccttga ctccccgggc tgtgtgcatt tcagacgggc tgtgctgaac actgcagctt 4560 gaatgagaat atcactgtcc cagacaccaa agttaatttc tatgcctgga agaggatgga 4620 ggtgagttcc tttttttttt tttttccttt cttttggaga atctcatttg cgagcctgat 4680 tttggatgaa agggagaatg atcgagggaa aggtaaaatg gagcagcaga gatgaggctg 4740 cctgggcgca gaggctcacg tctataatcc caggctgaga tggccgagat gggagaattg 4800 cttgagccct ggagtttcag accaacctag gcagcatagt gagatccccc atctctacaa 4860 acatttaaaa aaattagtca ggtgaagtgg tgcatggtgg tagtcccaga tatttggaag 4920 gctgaggcgg gaggatcgct tgagcccagg aatttgaggc tgcagtgagc tgtgatcaca 4980 ccactgcact ccagcctcag tgacagagtg aggccctgtc tcaaaaaaga aaagaaaaaa 5040 gaaaaataat gagggctgta tggaatacat tcattattca ttcactcact cactcactca 5100 ttcattcatt cattcattca acaagtctta ttgcatacct tctgtttgct cagcttggtg 5160 cttggggctg ctgaggggca ggagggagag ggtgacatgg gtcagctgac tcccagagtc 5220 cactccctgt aggtcgggca gcaggccgta gaagtctggc agggcctggc cctgctgtcg 5280 gaagctgtcc tgcggggcca ggccctgttg gtcaactctt cccagccgtg ggagcccctg 5340 cagctgcatg tggataaagc cgtcagtggc cttcgcagcc tcaccactct gcttcgggct 5400 ctgggagccc aggtgagtag gagcggacac ttctgcttgc cctttctgta agaaggggag 5460 aagggtcttg ctaaggagta caggaactgt ccgtattcct tccctttctg tggcactgca 5520 gcgacctcct gttttctcct tggcagaagg aagccatctc ccctccagat gcggcctcag 5580 ctgctccact ccgaacaatc actgctgaca ctttccgcaa actcttccga gtctactcca 5640 atttcctccg gggaaagctg aagctgtaca caggggaggc ctgcaggaca ggggacagat 5700 gacgtacaag taagaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 5760 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 5820 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 5880 cattgcaata gtgtgttgga attttttttg tctctcactc ggaaggacat atgggagggc 5940 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggatacat atgccatatg 6000 ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg 6060 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt 6120 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca 6180 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat 6240 ccctcgacct gcagcccaag cttgcatgcc tgcaggtcga ctctagtgga tcccccgccc 6300 cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat 6360 cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg 6420 gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg 6480 gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc 6540 cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 6600 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 6660 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 6720 gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg 6780 gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag 6840 cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg 6900 cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg 6960 gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg agcggatccg 7020 cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 7080 agcgattccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 7140 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 7200 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 7260 cggggccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 7320 aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 7380 gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 7440 gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 7500 tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 7560 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 7620 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 7680 gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg 7740 gatccgcggg gctgcaggaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 7800 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 7860 gcctaatgag tgagctaact cacattaatt gctttgcgct cactgcccgc tttccagtcg 7920 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 7980 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt ctttgggctg 8040 cggcgagcg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 8100 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 8160 gcgttgctgg cgtttttcca taggctccgc cCccctgacg agcatcacaa aaatcgacgc 8220 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 8280 agctccctcg tgcgctctcc tgttccgacc ctgcccctta ccggatacct gtccgccttt 8340 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 8400 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 8460 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 8520 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 8580 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 8640 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 8700 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 8760 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 8820 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 8880 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 8940 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9000 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9060 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9120 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9180 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9240 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9300 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9360 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 9420 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 9480 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 9540 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 9600 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 9660 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 9720 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 9780 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 9840 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 9900 acatttcccc gaaaagtgcc acctgacgta gttaacaaaa aaaagcccgc cgaagcgggc 9960 tttattacca agcgaagcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat 10020 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 10080 taagttgggt aaagccaggg ttttcccagt cacgacgttg taaaacgacg gccagtccgt 10140 aatacgactc acttaaggcc ttgactagag ggtcgacggt atacagacat gataagatac 10200 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 10260 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca acttggggtg 10320 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa 10380 aacgattccg aagcccaacc tttcatagaa ggcg cggtg gaatcgaaat ctcgtagcac 10440 gtgtcagtcc tgctcctcgg ccacgaagtg cacg 10474 <210> 126 <211> 6119 <212> DNA
<213> Artificial Sequence <220>
<223> pl8attBZeoeGFP Plasmid <400> 126 cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60 gtcatggccg gcccgaaggc gtcccggaag ttcgtggaca Cgacctccga ccactcggcg 120 tacagctcgt ccaggccgcg cacccacacc caagccaggg tgttgtccgg caccacctgg 180 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360 caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgggctg caggtcgagg 420 gatcttcata agagaagagg gacagctatg actgggagta gtcacgagag gaggaaaaat 480 ctggctagta aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa 540 caaaatataa aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca 600 gcagggggct gtttcatata ctgatgacct ctttatagcc acctttgttc atggcagcca 660 gcatatggca tatgttgcca aactctaaac caaatactca ttctgatgtt ttaaatgatt 720 tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca ctattgcaat 780 gaaaataaat ttcctttatt agccagaagt caaatgctca aggggcttca tgatgtcccc 840 ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg cattggccac 900 accagccacc accttctgat aggcagcctg cacctgagga gtgaattctt acttgtacag 960 ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc 1020 gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg 1080 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac 1140 gttgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt 1200 gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat 1260 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc 1320 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg 1380 ctcctggacg tagccttcgg gcatagcgga cttgaagaag tcgtgctgct tcatgtggtc 1440 ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg 1500 = ==.-cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc 1560 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac 1620 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggcgaattc 1680 tttgccaaaa tgatgagaca gcacaacaac cagcacgttg cccaggagct gtaggaaaaa 1740 gaagaaggca tgaacatggt tagCagaggc tctagagccg ccggtcacac gccagaagcc 1800 gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc ccctgcggca gccccgaggc 1860 tggagatgga gaaggggacg gcggcgcggc gacgcacgaa ggccctcccc gcccatttcc 1920 ttcctgccgg cgccgcaccg cttcgcccgc gcccgctaga gggggtgcgg cggcgcctcc 1980 cagatttcgg ctccgccaga tttgggacaa aggaagtccc tgcgccctct cgcacgatta 2040 ccataaaagg caatggctgc ggctcgccgc gcctcgacag ccgccggcgc tccggggccg 2100 ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc ccgccccgcc cggcaccccc 2160 acctgccgcc accccccgcc cggcacggcg agccccgcgc cacgccccgc acggagcccc 2220 gcacccgaag ccgggccgtg ctcagcaact cggggagggg ggtgcagggg ggggttacag 2280 cccgaccgcc gcgcccacac cccctgctca cccccccacg cacacacccc gcacgcagcc 2340 tttgttcccc tcgcagcccc cccgcaccgc ggggcaccgc ccccggccgc gctcccctcg 2400 cgcacacgcg gagcgcacaa agccccgcgc cgcgcccgca gcgctcacag ccgccgggca 2460 gccggggccg cacgcggcgc tccccacgca cacacacacg cacgcacccc ccgagccgct 2520 cccccccgca caaagggccc tcccggagcc ctttaaggct ttcacgcagc cacagaaaag 2580 aaacgagccg tcattaaacc aagcgctaat tacagcccgg aggagaaggg ccgtcccgcc 2640 cgctcacttg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc gaggcggcgc 2700 ggagcggggc acggggcgaa ggcaacgcag cgactcccgc ccgccgcgcg cttcgctttt 2760 tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct 2820 gattggctgc cgccgcacct ctccgcctcg.ccccgccccg cccctcgccc cgccccgccc 2880 cgcctggcgc gc9000CCcc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata 2940 aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg 3000 gggctcacct cgacccatgg taatagcgat gactaatacg tagatgtact gccaagtagg 3060 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac 3120 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta 3180 ccgtaaatag tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac 3240 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt 3300 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat 3360 tgattactat taataactag aggatccccg ggtaccgagc tcgaattcgt aatcatggtc 3420 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3480 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3540 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3600 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3660 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3720 atggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3780 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3840 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3900 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3960 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctggtagctc 4020 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4080 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4140 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4200 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4260 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4320 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4380 gattacccgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4440 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4500 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4560 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4620 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4680 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4740 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4800 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4860 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4920 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4980 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5040 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5100 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5160 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5220 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5280 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5340 atcttttact ttcaccagcg tttctgggtg aacaaaaaca ggaaggcaaa atgccgcaaa 5400 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5460 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5520 = =

aaataaacaa ataggggttc cgcgcacatt taccagaaaa gtgccacctg acgtagttaa 5580 caaaaaaaag cccgccgaag cgggctttat taccaagcga agcgccattc gccattcagg 5640 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 5700 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 5760 cgttgtaaaa cgacggccag tccgtaatac gactcactta aggccttgac tagagggtcg 5820 acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 5880 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5940 aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga 6000 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg 6060 cggtggaatc gaaatctcgt agcacgtgtc agtcctgctc ctcggccacg aagtgcacg 6119 <210> 127 <211> 5855 <212> DNA
<213> Artificial Sequence <220>
<223> pCXLamInt Plasmid (Wildtype Integrase) <400> 127 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atcggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccggc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgatctg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcggggcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620 gctctagagc ctctgctaac catgttcatg ccttcttctt tttccta.cag ctcctgggca 1680 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740 gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800 acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atggcaatca 1860 ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920 cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980 tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040 caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaatttcgg 2100 caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160 cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220 ctgccactcg cgcagcaaaa tcagaggtaa ggagatcaag acttacggct gacgaatacc 2280 tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340 ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400 atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460 tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520 ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580 caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640 cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700 ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760 gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820 S. .

cctatcagaa ggtggtggct ggtgtggcca atcccttgac tcacaaatac cactgagatc 2880 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060 gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt 3240 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300 gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600 tccgcccatt ctccgcccca tgcctgacta atttttttta tttatgcaga ggccgaggcc 3660 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720 tgcaaaaagc ttacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900 aggcggtttg ctgattggcc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4080 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tggtcacgct gtaggtatct 4320 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4380 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460 ttgctcttgc ccggcgtcaa tacgggataa taccgcccca catagcagaa ctttaaaagt 5520 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820 ggttccgcgc acatttcccc gaaaagtgcc acctg 5855 <210> 128 <211> 303 <212> DNA
<213> Artificial Sequence <220>
<223> Human FER-1 Promoter <400> 128 tccatgacaa agcacttttt gagcccaagc ccagcctagc tcgagctaaa cgggcacaga 60 gacgccaccg ctgtcccaga ggcagtcggc taccggtccc cgctcccgag ctccgccaga 120 gcgcgcgagg gcctccagcg gccgcccctc ccccacagca ggggcggggt cccgcgccca 180 ccggaaggag cgggctcggg gcgggcggcg ctgattggcc ggggcgggcc tgacgccgac 240 gcggctataa gagaccacaa gcgacccgca gggccagacg ttcttcgccg agagtcgggt 300 acc 303 <210> 129 <211> 6521 <212> DNA
<213> Artificial Sequence <220>
<223> pIRES-BSR Plasmid <400> 129 tcaatattgg ccattagcca tattattcat tgtttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720 agcagagctc gattagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080 ataggctagc ctcgagaatt cacgcgtcga gcatgcatct agggcggcca attccgcccc 1140 tctccctccc ccccccctaa cgttactggc caaagcccct tggaataagg ccggtgtgcg 1200 tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa 1260 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg 1320 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca 1380 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc 1440 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt 1500 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 1560 ctgaaggatg cccagaaggt accccattgt atggaatttg atctggggcc tcggtgcaca 1620 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac 1680 gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccaccatg aaaacattta 1740 acatttctca acaagatcta gaattagtag aagtagcgac agagaagatt acaatgcttt 1800 atgaggataa taaacatcat gtgggagcgg caattcgtac gaaaacagga gaaatcattt 1860 cggcagtaca tattgaagcg tatataggac gagtaactgt ttgtgcagaa gccattgcga 1920 ttggtagtgc agtttcgaat ggacaaaagg attttgacac gattgtagct gttagacacc 1980 cttattctga cgaagtagat agaagtattc gagtggtaag tccttgtggt atgtgtaggg 2040 agttgatttc agactatgca ccagattgtt ttgtgttaat agaaatgaat ggcaagttag 2100 tcaaaactac gattgaagaa ctcattccac tcaaatatac ccgaaattaa aagttttacc 2160 ataccaagct tggcgggcgg ccgcttccct ttagtgaggg ttaatgcttc gagcagacat 2220 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 2280 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 2340 agttaataac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 2400 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc cgataaggat cgatccgggc 2460 tggcgtaata gcgaataggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 2520 ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2580 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2640 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2700 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2760 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2820 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2880 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaa.tgag ctgatttaac 2940 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3000 tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat 3060 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 3120 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3180 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3240 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3300 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3360 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 3420 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca 3480 90 v1/097059 PCT/US02,, ,-52 caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg 3540 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3600 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3660 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3720 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3780 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3840 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3900 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3960 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4020 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4080 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 4140 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 4200 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 4260 ttctcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 4320 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc 4380 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg 4440 cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4500 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4560 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4620 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4680 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4740 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4800 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4860 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4920 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4980 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5040 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5100 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5160 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5220 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5280 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5340 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 5400 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5460 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5520 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5580 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5640 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5700 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5760 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5820 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5880 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5940 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6000 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6060 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtgggg ataagtcgtg 6120 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6180 gtggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6240 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6300 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 6360 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6420 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6480 ggccttttgc tggccttttg ctcacatggc tcgacagatc t 6521

Claims (4)

1. A lambda-integraseR mutein comprising an arginine at a position which corresponds to position 174 of the lambda-integraseR mutein as set forth in SEQ ID NO: 37.

2. A lambda-integraseR mutein, wherein the lambda-integraseR mutein comprises SEQ ID NO: 37.

3. The lambda-integraseR mutein of claim 1 or 2, wherein the mutein further comprises an amino acid signal for nuclear localization.

4. The lambda-integraseR mutein of any one of claims 1 to 3, wherein the mutein further comprises an epitope tag for protein purification.