AU2011253898A1 - Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries - Google Patents

Description

NOVEL METHODS OF CONSTRUCTING LIBRARIES COMPRISING DISPLAYED AND/OR EXPRESSED MEMBERS OF A DIVERSE FAMILY OF PEPTIDES, POLYPEPTIDES OR PROTEINS AND THE NOVEL LIBRARIES 5 RELATED APPLICATION AND TECHNICAL FIELD This application is a divisional of Application No. 2009200092, the entire content of which is incorpor-ated herein by cross-reference. The present invention relates to libraries 10 of genetic packages that display and/or express a member of a diverse family of peptides, polypeptides or proteins and collectively display and/or express at least a portion of the diversity of the family. In an alternative embodiment, the invention relates to 15 libraries that include a member of a diverse family of peptides, polypeptides or proteins and collect ively comprise at least a portion of the diversity of the family. In a preferred embodiment, the displayed and/or expressed polypeptides are human Fabs. 20 [Text continues on page 2.] -2 More specifically, the invention is directed to the methods of cleaving single-stranded nucleic acids at chosen locations, the cleaved nucleic acids encoding, at least in part, the peptides, polypeptides 5 or proteins displayed on the genetic packages of, and/or expressed in, the libraries of the invention. In a preferred embodiment, the genetic packages are filamentous phage or phagemids or yeast. The present invention further relates to 10 vectors for displaying and/or expressing a diverse family of peptides, polypeptides or proteins. The present invention further relates to methods of screening the libraries of the invention and to the peptides, polypeptides and proteins identified 15 by such screening. BACKGROUND OF THE INVENTION It is now common practice in the art to prepare libraries of genetic packages that display, express or comprise a member of a diverse family of 20 peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. In many common libraries, the peptides, polypeptides or proteins are related to antibodies. Often, they are Fabs or single chain 25 antibodies. In general, the DNAs that encode members of the families to be displayed and/or expressed must be amplified before they are cloned and used to display and/or express the desired member. Such amplification 30 typically makes use of forward and backward primers.

-3 Such primers can be complementary to sequences native to the DNA to be amplified or complementary to oligonucleotides attached at the 5' or 3' ends of that DNA. Primers that are complementary to 5 sequences native to the DNA to be amplified are disadvantaged in that they bias the members of the families to be displayed. Only those members that contain a sequence in the native DNA that is substantially complementary to the primer will be 10 amplified. Those that do not will be absent from the family. For those members that are amplified, any diversity within the primer region will be suppressed. For example, in European patent 368,684 B1, the primer that is used is at the 5' end of the VH 15 region of an antibody gene. It anneals to a sequence region in the native DNA that is said to be "sufficiently well conserved" within a single species. Such primer will bias the members amplified to those having this "conserved" region. Any diversity within 20 this region is extinguished. It is generally accepted that human antibody genes arise through a process that involves a combinatorial selection of V and J or V, D, and J followed by somatic mutations. Although most diversity 25 occurs in the Complementary Determining Regions (CDRs), diversity also occurs in the more conserved Framework Regions (FRs) and at least some of this diversity confers or enhances specific binding to antigens (Ag). As a consequence, libraries should contain as much of 30 the CDR and FR diversity as possible. To clone the amplified DNAs of the peptides, polypeptides or proteins that they encode for display on a genetic package and/or for expression, the DNAs -4 must be cleaved to produce appropriate ends for ligation to a vector. Such cleavage is generally effected using restriction endonuclease recognition sites carried on the primers. When the primers are at 5 the 5' end of DNA produced from reverse transcription of RNA, such restriction leaves deleterious 5' untranslated regions in the amplified DNA. These regions interfere with expression of the cloned genes and thus the display of the peptides, polypeptides and 10 proteins coded for by them. SUMMARY OF THE INVENTION It is an object of this invention to provide novel methods for constructing libraries that display, express or comprise a member of a diverse family of 15 peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. These methods are not biased toward DNAs that contain native sequences that are complementary to the primers used for amplification. 20 They also enable any sequences that may be deleterious to expression to be removed from the amplified DNA before cloning and displaying and/or expressing. It is another object of this invention to provide a method for cleaving single-stranded nucleic 25 acid sequences at a desired location, the method comprising the steps of: (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally 30 complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement -5 in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and 5 (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed 10 at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur 15 at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature. It is a further object of this invention to provide an alternative method for cleaving single 20 stranded nucleic acid sequences at a desired location, the method comprising the steps of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the 25 oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition 30 site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the -6 complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed 5 at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur 10 at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature. In an alternative embodiment of this object of the invention, the restriction endonuclease 15 recognition site is not initially located in the double-stranded part of the oligonucleotide. Instead, it is part of an amplification primer, which primer is complementary to the double-stranded region of the oligonucleotide. On amplification of the DNA-partially 20 double-stranded combination, the restriction endonuclease recognition site carried on the primer becomes part of the DNA. It can then be used to cleave the DNA. Preferably, the restriction endonuclease 25 recognition site is that of a Type II-S restriction endonuclease whose cleavage site is located at a known distance from its recognition site. It is another object of the present invention to provide a method of capturing DNA molecules that 30 comprise a member of a diverse family of DNAs and collectively comprise at least a portion of the diversity of the family. These DNA molecules in -7 single-stranded form have been cleaved by one of the methods of this invention. This method involves ligating the individual single-stranded DNA members of the family to a partially duplex DNA complex. The 5 method comprises the steps of: (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single 10 stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences 15 necessary to return the sequences that remain after cleavage into proper reading frame for expression and containing a restriction endonuclease recognition site 5' of those sequences; and 20 (ii) cleaving the partially double stranded oligonucleotide sequence solely at the restriction endonuclease cleavage site contained within the double-stranded region of the partially double-stranded 25 oligonucleotide. As before, in this object of the invention, the restriction endonuclease recognition site need not be located in the double-stranded portion of the oligonucleotide. Instead, it can be introduced on 30 amplification with an amplification primer that is used to amplify the DNA-partially double-stranded oligonucleotide combination.

- 8 It is another object of this invention to prepare libraries, that display, express or comprise a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least part of the diversity of the family, using the methods and 3 DNAs described above. It is an object of this invention to screen those libraries to identify useful peptides, polypeptides and proteins and to use those substances in human therapy. Additional objects of the invention are reflected in the claims. ) Each of these claims is specifically incorporated by reference in this specification. A definition of the specific embodiment of the invention claimed herein follows. In a broad format, the invention provides a method for cleaving 5 a nucleic acid at a desired location, the method comprising the steps of: (i) contacting a single-stranded nucleic acid with a single stranded oligonucleotide, the single-stranded oligonucleotide being complementary to the single-stranded nucleic acid in the 0 region in which cleavage is desired; wherein the single-stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the locally double-stranded region com prises a restriction endonuclease recognition site; and 25 (ii) cleaving the nucleic acid at the restriction endonuclease recognition, wherein the cleaving comprises contacting a restriction endonuclease to the locally double-stranded region, wherein the restriction endonuclease is specific for the restriction endonuclease recognition site; 30 the contacting and the cleaving steps being performed at a tempera ture wherein the single-stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the remainder of the single-stranded nucleic acid is single-stranded, and wherein the 35 restriction endonuclease is active at the temperature.

- 8a BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of various methods that may be employed to amplify VH genes without using primers specific for VH sequences. FIG. 2 is a schematic of various methods that may be employed to amplify VL genes without using primers specific for VL sequences. FIG. 3 is a schematic of RACE amplification of antibody heavy and light chains. FIG. 4 depicts gel analysis of amplification products obtained after the primary PCR reaction from 4 different patient samples. D FIG. 5 depicts gel analysis of cleaved kappa DNA from Example 2. FIG. 6 depicts gel analysis of extender-cleaved kappa DNA from Example 2. [Text continues on page 9.] -9 FIG. 7 depicts gel analysis of the PCR product from the extender-kappa amplification from Example 2. FIG. 8 depicts gel analysis of purified PCR 5 product from the extender-kappa amplification from Example 2. FIG. 9 depicts gel analysis of cleaved and ligated kappa light chains from Example 2. FIG. 10 is a schematic of the design for CDR1 10 and CDR2 synthetic diversity. FIG. 11 is a schemaitc of the cloning schedule for construction of the heavy chain repertoire. FIG. 12 is a schematic of the cleavage and 15 ligation of the antibody light chain. FIG. 13 depicts gel analysis of cleaved and ligated lambda light chains from Example 4. FIG. 14 is a schematic of the cleavage and ligation of the antibody heavy chain. 20 FIG. 15 depicts gel analysis of cleaved and ligated lambda light chains from Example 5. FIG. 16 is a schematic of a phage display vector. FIG. 17 is a schematic of a Fab cassette. 25 FIG. 18 is a schematic of a process for incorporating fixed FRl residues in an antibody lambda sequence. FIG. 19 is a schematic of a process for incorporating fixed FR1 residues in an antibody kappa 30 sequence. FIG. 20 is a schematic of a process for incorporating fixed FR1 residues in an antibody heavy chain sequence.

- 10 TERMS In this application, the following terms and abbreviations are used: Sense strand The upper strand of ds DNA as 5 usually written. In the sense strand, 5'-ATG-3' codes for Met. Antisense strand The lower strand of ds DNA as usually written. In the 10 antisense strand, 3'-TAC-5' would correspond to a Met codon in the sense strand. Forward primer A "forward" primer is complementary to a part of the 15 sense strand and primes for synthesis of a new antisense strand molecule. "Forward primer" and "lower-strand primer" are equivalent. 20 Backward primer A "backward" primer is complementary to a part of the antisense strand and primes for synthesis of a new sense strand molecule. "Backward 25 primer" and "top-strand primer" are equivalent.

- 11 Bases Bases are specified either by their position in a vector or gene as their position within a gene by codon and base. For 5 example, "89.1" is the first base of codon 89, 89.2 is the second base of codon 89. Sv Streptavidin Ap Ampicillin 10 apR A gene conferring ampicillin resistance. RERS Restriction endonuclease recognition site RE Restriction endonuclease 15 cleaves preferentially at RERS URE Universal restriction endonuclease Functionally complementary Two sequences are sufficiently 20 complementary so as to anneal under the chosen conditions. AA Amino acid PCR Polymerization chain reaction - 12 GLGs Germline genes Ab Antibody: an immunoglobin. The term also covers any protein having a binding 5 domain which is homologous to an immunoglobin binding domain. A few examples of antibodies within this definition are, inter alia, 10 immunoglobin isotypes and the Fab, F(abl)2, scfv, Fv, dAb and Fd fragments. Fab Two chain molecule comprising an Ab light chain and part of 15 a heavy-chain. scFv A single-chain Ab comprising either VH::linker::VL or VL::linker::VH w.t. Wild type 20 HC Heavy chain LC Light chain VK A variable domain of a Kappa light chain. VH A variable domain of a heavy 25 chain.

- 13 VL A variable domain of a lambda light chain. In this application when it is said that nucleic acids are cleaved solely at the cleavage site 5 of a restriction endonuclease, it should be understood that minor cleavage may occur at random, e.g., at non specific sites other than the specific cleavage site that is characteristic of the restriction endonuclease. The skilled worker will recognize that such non 10 specific, random cleavage is the usual occurrence. Accordingly, "solely at the cleavage site" of a restriction endonuclease means that cleavage occurs preferentially at the site characteristic of that endonuclease. 15 As used in this application and claims, the term "cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide" includes cleavage sites formed by the single-stranded portion of the partially double 20 stranded ologonucleotide duplexing with the single stranded DNA, cleavage sites in the double-stranded portion of the partially double-stranded oligonucleotide, and cleavage sites introduced by the amplification primer used to amplify the single 25 stranded DNA-partially double-stranded oligonucleotide combination. In the two methods of this invention for preparing single-stranded nucleic acid sequences, the first of those cleavage sites is preferred. In the 30 methods of this invention for capturing diversity and cloning a family of diverse nucleic acid sequences, the latter two cleavage sites are preferred.

- 14 In this application, all references referred to are specifically incorporated by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The nucleic acid sequences that are useful in 5 the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins displayed, or expressed in or comprising the libraries of this invention, may be native, synthetic or a combination thereof. They may be mRNA, 10 DNA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies. Most preferably, they encode Fabs. The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be 15 introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes. Preferably, it is introduced into CDR1 and CDR2 of immunoglobulins. Preferably, 20 natural diversity is captured in the CDR3 regions of the immunoglogin genes of this invention from B cells. Most preferably, the nucleic acids of this invention comprise a population of immunoglobin genes that comprise synthetic diversity in at least one, and more 25 preferably both of the CDRl and CDR2 and diversity in CDR3 captured from B cells. Synthetic diversity may be created, for example, through the use of TRIM technology (U.S. 5,869,644). TRIM technology allows control over 30 exactly which amino-acid types are allowed at variegated positions and in what proportions. In TRIM technology, codons to be diversified are synthesized - 15 using mixtures of trinucleotides. This allows any set of amino acid types to be included in any proportion. Another alternative that may be used to generate diversified DNA is mixed oligonucleotide 5 synthesis. With TRIM technology, one could allow Ala and Trp. With mixed oligonucleotide synthesis, a mixture that included Ala and Trp would also necessarily include Ser and Gly. The amino-acid types allowed at the variegated positions are picked with 10 reference to the structure of antibodies, or other peptides, polypeptides or proteins of the family, the observed diversity in germline genes, the observed somatic mutations frequently observed, and the desired areas and types of variegation. 15 In a preferred embodiment of this invention, the nucleic acid sequences for at least one CDR or other region of the peptides, polypeptides or proteins of the family are cDNAs produced by reverse transcription from mRNA. More preferably, the mRNAs 20 are obtained from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells (such as B-lymphocytes or plasma cells) that express members of naturally diverse sets of related genes. More preferable, the mRNAs encode a diverse family of 25 antibodies. Most preferably, the mRNAs are obtained from patients suffering from at least one autoimmune disorder or cancer. Preferably, mRNAs containing a high diversity of autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid 30 arthritis, antiphospholipid syndrome and vasculitis are used. In a preferred embodiment of this invention, the cDNAs are produced from the mRNAs using reverse - 16 transcription. In this preferred embodiment, the mRNAs are separated from the cell and degraded using standard methods, such that only the full length (i.e., capped) mRNAs remain. The cap is then removed and reverse 5 transcription used to produce the cDNAs. The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., HJ de Haard et al., Journal of Biological Chemistry, 274 (26) :18218-30 10 (1999). In the preferred embodiment of this invention where the mRNAs encode antibodies, primers that are complementary to the constant regions of antibody genes may be used. Those primers are useful because they do not generate bias toward subclasses of antibodies. In 15 another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes). Alternatively, sequences complementary to the primer may be attached to the termini of the antisense strand. In one preferred embodiment of this 20 invention, the reverse transcriptase primer may be biotinylated, thus allowing the cDNA product to be immobilized on streptavidin (Sv) beads. Immobilization can also be effected using a primer labeled at the 5' end with one of a) free amine group, b) thiol, c) 25 carboxylic acid, or d) another group not found in DNA that can react to form a strong bond to a known partner on an insoluble medium. If, for example, a free amine (preferably primary amine) is provided at the 5' end of a DNA primer, this amine can be reacted with carboxylic 30 acid groups on a polymer bead using standard amide forming chemistry. If such preferred immobilization is used during reverse transcription, the top strand RNA is degraded using well-known enzymes, such as a - 17 combination of RNAseH and RNAseA, either before or after immobilization. The nucleic acid sequences useful in the methods of this invention are generally amplified 5 before being used to display and/or express the peptides, polypeptides or proteins that they encode. Prior to amplification, the single-stranded DNAs may be cleaved using either of the methods described before. Alternatively, the single-stranded DNAs may be 10 amplified and then cleaved using one of those methods. Any of the well known methods for amplifying nucleic acid sequences may be used for such amplification. Methods that maximize, and do not bias, diversity are preferred. In a preferred embodiment of 15 this invention where the nucleic acid sequences are derived from antibody genes, the present invention preferably utilizes primers in the constant regions of the heavy and light chain genes and primers to a synthetic sequence that are attached at the 5' end of 20 the sense strand. Priming at such synthetic sequence avoids the use of sequences within the variable regions of the antibody genes. Those variable region priming sites generate bias against V genes that are either of rare subclasses or that have been mutated at the 25 priming sites'. This bias is partly due to suppression of diversity within the primer region and partly due to lack of priming when many mutations are present in the region complementary to the primer. The methods disclosed in this invention have the advantage of not 30 biasing the population of amplified antibody genes for particular V gene types. The synthetic sequences may be attached to the 5' end of the DNA strand by various methods well - 18 known for ligating DNA sequences together. RT CapExtention is one preferred method. In RT CapExtention (derived from Smart PCR (TM), a short overlap (5'-...GGG-3' in the upper 5 strand primer (USP-GGG) complements 3'-CCC.... 5' in the lower strand) and reverse transcriptases are used so that the reverse complement of the upper-strand primer is attached to the lower strand. FIGs. 1 and 2 show schematics to amplify VH 10 and VL genes using RT CapExtention. FIG. 1 shows a schematic of the amplification of VH genes. FIG. 1, Panel A shows a primer specific to the poly-dT region of the 3' UTR priming synthesis of the first, lower strand. Primers that bind in the constant region are 15 also suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3' terminal CCCs and extending the 20 reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that replicates the 5' end of the synthetic primer of panel 25 C and a bottom-strand primer complementary to part of the constant domain. Panel E shows immobilized double stranded (ds) cDNA obtained by using a 5'-biotinylated top-strand primer. FIG. 2 shows a similar schematic for 30 amplification of VL genes. FIG. 2, Panel A shows a primer specific to the constant region at or near the 3' end priming synthesis of the first, lower strand. Primers that bind in the poly-dT region are also - 19 suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that 5 hybridize to the 3' terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that 10 replicates the 5' end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. The bottom-strand primer also contains a useful restriction endonuclease site, such as AscI. Panel E shows immobilized ds cDNA obtained by 15 using a 5'-biotinylated top-strand primer. In FIGs. 1 and 2, each V gene consists of a 5' untranslated region (UTR) and a secretion signal, followed by the variable region, followed by a constant region, followed by a 3' untranslated region (which 20 typically ends in poly-A). An initial primer for reverse transcription may be complementary to the constant region or to the poly A segment of the 3'-UTR. For human heavy-chain genes, a primer of 15 T is preferred. Reverse transcriptases attach several C 25 residues to the 3' end of the newly synthesized DNA. RT CapExtention exploits this feature. The reverse transcription reaction is first run with only a lower strand primer. After about 1 hour, a primer ending in GGG (USP-GGG) and more RTase are added. This causes 30 the lower-strand cDNA to be extended by the reverse complement of the USP-GGG up to the final GGG. Using one primer identical to part of the attached synthetic sequence and a second primer complementary to a region - 20 of known sequence at the 3' end of the sense strand, all the V genes are amplified irrespective of their V gene subclass. In another preferred embodiment, synthetic 5 sequences may be added by Rapid Amplification of cDNA Ends (RACE) (see Frohman, M.A., Dush, M.K., & Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA (85): 8998-9002). FIG. 1 shows a schematic of RACE 10 amplification of antibody heavy and light chains. First, mRNA is selected by treating total or poly(A+) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules that have them such as ribosomal RNA, fragmented mRNA, tRNA and genomic 15 DNA. Full length mRNA (containing a protective 7 methyl cap structure) is uneffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from full length mRNAs leaving a 5'-monophosphate group. Next, a synthetic RNA 20 adaptor is ligated to the RNA population, only molecules which have a 5-phosphate (uncapped, full length mRNAs) will accept the adaptor. Reverse trascriptase reactions using an oligodT primer, and nested PCR (using one adaptor primer (located in the 5' 25 synthetic adaptor) and one primer for the gene) are then used to amplify the desired transcript. In a preferred embodiment of this invention, the upper strand or lower strand primer may be also biotinylated or labeled at the 5' end with one of a) 30 free amino group, b) thiol, c) carboxylic acid and d) another group not found in DNA that can react to form a strong bond to a known partner as an insoluble medium. These can then be used to immobilize the labeled strand - 21 after amplification. The immobilized DNA can be either single or double-stranded. After amplification (using e.g., RT CapExtension or RACE), the DNAs of this invention are 5 rendered single-stranded. For example, the strands can be separated by using a biotinylated primer, capturing the biotinylated product on streptavidin beads, denaturing the DNA, and washing away the complementary strand. Depending on which end of the captured DNA is 10 wanted, one will choose to immobilize either the upper (sense) strand or the lower (antisense) strand. To prepare the single-stranded amplified DNAs for cloning into genetic packages so as to effect display of, or for expression of, the peptides, 15 polypeptides or proteins encoded, at least in part, by those DNAs, they must be manipulated to provide ends suitable for cloning and display and/or expression. In particular, any 5' untranslated regions and mammalian signal sequences must be removed and replaced, in 20 frame, by a suitable signal sequence that functions in the display or expression host. Additionally, parts of the variable domains (in antibody genes) may be removed and replaced by synthetic segments containing synthetic diversity. The diversity of other gene families may 25 likewise be expanded with synthetic diversity. According to the methods of this invention, there are two ways to manipulate the single-stranded DNAs for display and/or expression. The first method comprises the steps of: 30 (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the - 22 region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on 5 restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the 10 oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the 15 nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature. 20 In this first method, short oligonucleotides are annealed to the single-stranded DNA so that restriction endonuclease recognition sites formed within the now locally double-stranded regions of the DNA can be cleaved. In particular, a recognition site 25 that occurs at the same position in a substantial fraction of the single-stranded DNAs is identical. For antibody genes, this can be done using a catalog of germline sequences. See, e.g., "http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.htm 30 1." Updates can be obtained from this site under the heading "Amino acid and nucleotide sequence alignments." For other families, similar comparisons - 23 exist and may be used to select appropriate regions for cleavage and to maintain diversity. For example, Table 1 depicts the DNA sequences of the FR3 regions of the 51 known human VH 5 germline genes. In this region, the genes contain restriction endonuclease recognition sites shown in Table 2. Restriction endonucleases that cleave a large fraction of germline genes at the same site are preferred over endonucleases that cut at a variety of 10 sites. Furthermore, it is preferred that there be only one site for the restriction endonucleases within the region to which the short oligonucleotide binds on the single-stranded DNA, e.g., about 10 bases on either side of the restriction endonuclease recognition site. 15 An enzyme that cleaves downstream in FR3 is also more preferable because it captures fewer mutations in the framework. This may be advantageous is some cases. However, it is well known that framework mutations exist and confer and enhance 20 antibody binding. The present invention, by choice of appropriate restriction site, allows all or part of FR3 diversity to be captured. Hence, the method also allows extensive diversity to be captured. Finally, in the methods of this invention 25 restriction endonucleases that are active between about 37*C and about 75*C are used. Preferably, restriction endonucleases that are active between about 45 0 C and about 75 0 C may be used. More preferably, enzymes that are active above 50 0 C, and most preferably active about 30 55 0 C, are used. Such temperatures maintain the nucleic acid sequence to be cleaved in substantially single stranded form.

- 24 Enzymes shown in Table 2 that cut many of the heavy chain FR3 germline genes at a single position include: MaeIII(24@4), Tsp45I(21@4), HphI(44@5), BsaJI(23@65), AluI(23@47), BlpI(21@48), DdeI(29@58), 5 BglII(10@61), MslI(44@72), BsiEI(23@74), EaeI(23@74), EagI(23@74), HaeIII(25@75), Bst4CI(51@86), HpyCH4111(51@86), HinfI(38@2), MlyI(18@2), PleI(18@2), MnlI(31@67), HpyCH4V(21@44), BsmAI(16@ll), BpmI(19@12), XmnI(12@30), and SacI(ll@51). (The notation used 10 means, for example, that BsmAI cuts 16 of the FR3 germline genes with a restriction endonuclease recognition site beginning at base 11 of FR3.) For cleavage of human heavy chains in FR3, the preferred restriction endonucleases are: Bst4CI (or 15 TaaI or HpyCH4III), BlpI, HpyCH4V, and Ms1. Because ACNGT (the restriction endonuclease recognition site for Bst4CI, TaaI, and HpyCH411I) is found at a consistent site in all the human FR3 germline genes, one of those enzymes is the most preferred for capture 20 of heavy chain CDR3 diversity. BlpI and HpyCH4V are complementary. BlpI cuts most members of the VH1 and VH4 families while HpyCH4V cuts most members of the VH3, VH5, VH6, and VH7 families. Neither enzyme cuts VH2s, but this is a very small family, containing only 25 three members. Thus, these enzymes may also be used in preferred embodiments of the methods of this invention. The restriction endonucleases HpyCH4III, Bst4CI, and TaaI all recognize 5'-ACnGT-3' and cut upper strand DNA after n and lower strand DNA before 30 the base complementary to n. This is the most preferred restriction endonuclease recognition site for this method on human heavy chains because it is found in all germline genes. Furthermore, the restriction - 25 endonuclease recognition region (ACnGT) matches the second and third bases of a tyrosine codon (tay) and the following cysteine codon (tgy) as shown in Table 3. These codons are highly conserved, especially the 5 cysteine in mature antibody genes. Table 4 E shows the distinct oligonucleotides of length 22 (except the last one which is of length 20) bases. Table 5 C shows the analysis of 1617 actual heavy chain antibody genes. Of these, 1511 have the 10 site and match one of the candidate oligonucleotides to within 4 mismatches. Eight oligonucleotides account for most of the matches and are given in Table 4 F.l. The 8 oligonucleotides are very similar so that it is likely that satisfactory cleavage will be achieved with 15 only one oligonucleotide (such as H43.77.97.1-02#1) by adjusting temperature, pH, salinity, and the like. One or two oligonucleotides may likewise suffice whenever the germline gene sequences differ very little and especially if they differ very little close to the 20 restriction endonuclease recognition region to be cleaved. Table 5 D shows a repeat analysis of 1617 actual heavy chain antibody genes using only the 8 chosen oligonucleotides. This shows that 1463 of the sequences match at least one of the oligonucleotides to 25 within 4 mismatches and have the site as expected. Only 7 sequences have a second HpyCH4III restriction endonuclease recognition region in this region. Another illustration of choosing an appropriate restriction endonuclease recognition site 30 involves cleavage in FRl of human heavy chains. Cleavage in FRl allows capture of the entire CDR diversity of the heavy chain.

- 26 The germline genes for human heavy chain FR1 are shown in Table 6. Table 7 shows the restriction endonuclease recognition sites found in human germline genes FRIs. The preferred sites are BsgI(GTGCAG;39@4), 5 BsoFI(GCngc;43@6,11@9,2@3,1@12), TseI(Gcwgc;43@6,11@9,2@3,1@12), MspA1I(CMGckg;46@7,2@1), PvuII(CAGctg;46@7,2@1), AluI(AGct;48@82@2), DdeI(Ctnag;22@52,9@48), HphI(tcacc;22@80), BssKI(Nccngg;35@39,2@40), 10 BsaJI(Ccnngg;32@40,2@41), RstNT(CCwgg;33@40), ScrFI(CCngg;35@40,2@41), EcoOl9I(RGgnccy;22@46, 11@43), Sau96I(Ggncc;23@47,11@44), AvaII(Ggwcc;23@47,4@44), PpuMI(RGgwccy;22@46,4@43), BsmFI(gtccc;20@48), HinfI(Gantc;34@16,21@56,21@77), 15 TfiI(21@77), MlyI(GAGTC;34@16), MlyI(gactc;21@56), and AlwNI(CAGnnnctg;22@68). The more preferred sites are MspAI and PvuII. MspAI and PvuII have 46 sites at 7-12 and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are used that do not fully cover the 20 site at 1-6. Thus, the DNA will not be cleaved at that site. We have shown that DNA that extends 3, 4, or 5 bases beyond a PvuII-site can be cleaved efficiently. Another illustration of choosing an appropriate restriction endonuclease recognition site 25 involves cleavage in FRl of human kappa light chains. Table 8 shows the human kappa FRl germline genes and Table 9 shows restriction endonuclease recognition sites that are found in a substantial number of human kappa FR1 germline genes at consistent locations. Of 30 the restriction endonuclease recognition sites listed, BsmAI and Pf1FI are the most preferred enzymes. BsmAI sites are found at base 18 in 35 of 40 germl'ine genes.

- 27 PflFI sites are found in 35 of 40 germline genes at base 12. Another example of choosing an appropriate restriction endonuclease recognition site involves 5 cleavage in FR1 of the human lambda light chain. Table 10 shows the 31 known human lambda FR1 germline gene sequences. Table 11 shows restriction endonuclease recognition sites found in human lambda FR1'germline genes. HinfI and DdeI are the most preferred 10 restriction endonucleases for cutting human lambda chains in FR1. After the appropriate site or sites for cleavage are chosen, one or more short oligonucleotides are prepared so as to functionally complement, alone or 15 in combination, the chosen recognition site. The oligonucleotides also include sequences that flank the recognition site in the majority of the amplified genes. This flanking region allows the sequence to anneal to the single-stranded DNA sufficiently to allow 20 cleavage by the restriction endonuclease specific for the site chosen. The actual length and sequence of the oligonucleotide depends on the recognition site and the conditions to be used for contacting and cleavage. The 25 length must be sufficient so that the oligonucleotide is functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location. 30 Typically, the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, annealing is too weak and above 30 bases there can be a - 28 loss of specificity. A preferred length is 18 to 24 bases. Oligonucleotides of this length need not be identical complements of the germline genes. Rather, a 5 few mismatches taken may be tolerated. Preferably, however, no more than 1-3 mismatches are allowed. Such mismatches do not adversely affect annealing of the oligonucleotide to the single-stranded DNA. Hence, the two DNAs are said to be functionally complementary. 10 The second method to manipulate the single stranded DNAs of this invention for display and/or expression comprises the steps of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, 15 the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide 20 having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the 25 single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the 30 oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur - 29 at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature. As explained above, the cleavage site may be 5 formed by the single-stranded portion of the partially double-stranded oligonucleotide duplexing with the single-stranded DNA, the cleavage site may be carried in the double-stranded portion of the partially double stranded oligonucleotide, or the cleavage site may be 10 introduced by the amplification primer used to amplify the single-stranded DNA-partially double-stranded oligonucleotide combination. In this embodiment, the first is preferred. And, the restriction endonuclease recognition site may be located in either the double 15 stranded portion of the oligonucleotide or introduced by the amplification primer, which is complementary to that double-stranded region, as used to amplify the combination. Preferably, the restriction endonuclease site 20 is that of a Type II-S restriction endonuclease, whose cleavage site is located at a known distance from its recognition site. This second method, preferably, employs Universal Restriction Endonucleases ("URE"). UREs are 25 partially double-stranded oligonucleotides. The single-stranded portion or overlap of the URE consists of a DNA adapter that is functionally complementary to the sequence to be cleaved in the single-stranded DNA. The double-stranded portion consists of a restriction 30 endonuclease recognition site, preferably type II-S. The URE method of this invention is specific and precise and can tolerate some (e.g., 1-3) mismatches in the complementary regions, i.e., it is - 30 functionally complementary to that region. Further, conditions under which the URE is used can be adjusted so that most of the genes that are amplified can be cut, reducing bias in the library produced from those 5 genes. The sequence of the single-stranded DNA adapter or overlap portion of the URE typically consists of about 14-22 bases. However, longer or shorter adapters may be used. The size depends on the 10 ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used for contacting the URE and the single stranded DNA at the temperature used for cleaving the DNA with the restriction enzyme. The adapter must be 15 functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that the cleavage may occur at the chosen temperature and at the desired location. We prefer singe-stranded or overlap portions of 14-17 20 bases in length, and more preferably 18-20 bases in length. The site chosen for cleavage using the URE is preferably one that is substantially conserved in the family of amplified DNAs. As compared to the first 25 cleavage method of this invention, these sites do not need to be endonuclease recognition sites. However, like the first method, the sites chosen can be synthetic rather than existing in the native DNA. Such sites may be chosen by references to the -sequences of 30 known antibodies or other families of genes. For example, the sequences of many germline genes are reported at http://www.mrc-cpe.cam.ac.uk/imt doc/restricted/ok.html. For example, one preferred - 31 site occurs near the end of FR3 -- codon 89 through the second base of codon 93. CDR3 begins at codon 95. The sequences of 79 human heavy-chain genes are also available at 5 http://www.ncbi.nlm.nih.gov/entre2/nucleotide.html. This site can be used to identify appropriate sequences for URE cleavage according to the methods of this invention. See, e.g., Table 12B. Most preferably, one or more sequences are 10 identified using these sites or other available sequence information. These sequences together are present in a substantial fraction of the amplified DNAs. For example, multiple sequences could be used to allow for known diversity in germline genes or for 15 frequent somatic mutations. Synthetic degenerate sequences could also be used. Preferably, a sequence(s) that occurs in at least 65% of genes examined with no more than 2-3 mismatches is chosen URE single-stranded adapters or overlaps are 20 then made to be complementary to the chosen regions. Conditions for using the UREs are determined empirically. These conditions should allow cleavage of DNA that contains the functionally complementary sequences with no more than 2 or 3 mismatches but that 25 do not allow cleavage of DNA lacking such sequences. As described above, the double-stranded portion of the URE includes an endonuclease recognition site, preferably a Type II-S recognition site. Any enzyme that is active at a temperature necessary to 30 maintain the single-stranded DNA substantially in that form and to allow the single-stranded DNA adapter portion of the URE to anneal long enough to the single- - 32 stranded DNA to permit cleavage at the desired site may be used. The preferred Type II-S enzymes for use in the URE methods of this invention provide asymmetrical 5 cleavage of the single-stranded DNA. Among these are the enzymes listed in Table 13. The most preferred Type II-S enzyme is FokI. When the preferred FokI containing URE is used, several conditions are preferably used to effect 10 cleavage: 1) Excess of the URE over target DNA should be present to activate the enzyme. URE present only in equimolar amounts to the target DNA would yield poor cleavage of ssDNA because 15 the amount of active enzyme available would be limiting. 2) An activator may be used to activate part of the FokI enzyme to dimerize without causing cleavage. Examples of appropriate activators 20 are shown in Table 14. 3) The cleavage reaction is performed at a temperature between 45*-75 0 C, preferably above 50 0 C and most preferably above 55*C. The UREs used in the prior art contained a 25 14-base single-stranded segment, a 10-base stem (containing a FokI site), followed by the palindrome of the 10-base stem. While such UREs may be used in the methods of this invention, the preferred UREs of this invention also include a segment of three to eight 30 bases (a loop) between the FokI restriction endonuclease recognition site containing segments. In the preferred embodiment, the stem (containing the FokI - 33 site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these "lollipop" URE adapters are shown in Table 15. One example of using a URE to cleave an 5 single-stranded DNA involves the FR3 region of human heavy chain. Table 16 shows an analysis of 840 full length mature human heavy chains with the URE recognition sequences shown. The vast majority (718/840=0.85) will be recognized with 2 or fewer 10 mismatches using five UREs (VHS881-1.1, VHS881-1.2, VHS881-2.1, VHS881-4.1, and VHS881-9.1). Each has a 20-base adaptor sequence to complement the germline gene, a ten-base stem segment containing a FokI site, a five base loop, and the reverse complement of the first 15 stem segment. Annealing those adapters, alone or in combination, to single-stranded antisense heavy chain DNA and treating with FokI in the presence of, e.g., the activator FOKIact, will lead to cleavage of the antisense strand at the position indicated. 20 Another example of using a URE(s) to cleave a single-stranded DNA involves the FR1 region of the human Kappa light chains. Table 17 shows an analysis of 182 full-length human kappa chains for matching by the four 19-base probe sequences shown. Ninety-six 25 percent of the sequences match one of the probes with 2 or fewer mismatches. The URE adapters shown in Table 17 are for cleavage of the sense strand of kappa chains. Thus, the adaptor sequences are the reverse complement of the germline gene sequences. The URE 30 consists of a ten-base stem, a five base loop, the reverse complement of the stem and the complementation sequence. The loop shown here is TTGTT, but other sequences could be used. Its function is to interrupt - 34 the palindrome of the stems so that formation of a lollypop monomer is favored over dimerization. Table 17 also shows where the sense strand is cleaved. Another example of using a URE to cleave a 5 single-stranded DNA involves the human lambda light chain. Table 18 shows analysis of 128 human lambda light chains for matching the four 19-base probes shown. With three or fewer mismatches, 88 of 128 (69%) of the chains match one of the probes. Table 18 also 10 shows URE adapters corresponding to these probes. Annealing these adapters to upper-strand ssDNA of lambda chains and treatment with FokI in the presence of FOKIact at a temperature at or above 45*C will lead to specific and precise cleavage of the chains. 15 The conditions under which the short oligonucleotide sequences of the first method and the UREs of the second method are contacted with the single-stranded DNAs may be empirically determined. The conditions must be such that the single-stranded 20 DNA remains in substantially single-stranded form. More particularly, the conditions must be such that the single-stranded DNA does not form loops that may int-rfere with its association with the oligonucleotide sequence or the URE or that may themselves provide 25 sites for cleavage by the chosen restriction endonuclease. The effectiveness and specificity of short oligonucleotides (first method) and UREs (second method) can be adjusted by controlling the 30 concentrations of the URE adapters/oligonucleotides and substrate DNA, the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and - 35 formamide), the concentration of the restriction endonuclease(e.g., FokI), and the time of the digestion. These conditions can be optimized with synthetic oligonucleotides having: 1) target germline 5 gene sequences, 2) mutated target gene sequences, or 3) somewhat related non-target sequences. The goal is to cleave most of the target sequences and minimal amounts of non-targets. In accordance with this invention, the 10 single-stranded DNA is maintained in substantially that form using a temperature between about 370C and about 750C. Preferably, a temperature between about 45*C and about 750C is used. More preferably, a temperature between 500C and 600C, most preferably between 550C and 15 600C, is used. These temperatures are employed both when contacting the DNA with the oligonucleotide or URE and when cleaving the DNA using the methods of this invention. The two cleavage methods of this invention 20 have several advantages. The first method allows the individual members of the family of single-stranded DNAs to be cleaved preferentially at one substantially conserved endonuclease recognition site. The method also does not require an endonuclease recognition site 25 to be built into the reverse transcription or amplification primers. Any native or synthetic site in the family can be used. The second method has both of these advantages. In addition, the preferred URE method 30 allows the single-stranded DNAs to be cleaved at positions where no endonuclease recognition site naturally occurs or has been synthetically constructed.

- 36 Most importantly, both cleavage methods permit the use of 5' and 3' primers so as to maximize diversity and then cleavage to remove unwanted or deleterious sequences before cloning, display and/or 5 expression. After cleavage of the amplified DNAs using one of the methods of this invention, the DNA is prepared for cloning, display and/or expression. This is done by using a partially duplexed synthetic DNA 10 adapter, whose terminal sequence is based on the specific cleavage site at which the amplified DNA has been cleaved. The synthetic DNA is designed such that when it is ligated to the cleaved single-stranded DNA in 15 proper reading frame so that the desired peptide, polypeptide or protein can be displayed on the surface of the genetic package and/or expressed. Preferably, the double-stranded portion of the adapter comprises the sequence of several codons that encode the amino 20 acid sequence characteristic of the family of peptides, polypeptides or proteins up to the cleavage site. For human heavy chains, the amino acids of the 3-23 framework are preferably used to provide the sequences required for expression of the cleaved DNA. 25 Preferably, the double-stranded portion of the adapter is about 12 to 100 bases in length. More preferably, about 20 to 100 bases are used. The double-standard region of the adapter also preferably contains at least one endonuclease recognition site 30 useful for cloning the DNA into a suitable display and/or expression vector (or a recipient vector used to archive the diversity). This endonuclease restriction site may be native to the germline gene sequences used - 37 to extend the DNA sequence. It may be also constructed using degenerate sequences to the native germline gene sequences. Or, it may be wholly synthetic. The single-stranded portion of the adapter is 5 complementary to the region of the cleavage in the single-stranded DNA. The overlap can be from about 2 bases up to about 15 bases. The longer the overlap, the more efficient the ligation is likely to be. A preferred length for the overlap is 7 to 10. This 10 allows some mismatches in the region so that diversity in this region may be captured. The single-stranded region or overlap of the partially duplexed adapter is advantageous because it allows DNA cleaved at the chosen site, but not other 15 fragments to be captured. Such fragments would contaminate the library with genes encoding sequences that will not fold into proper antibodies and are likely to be non-specifically sticky. One illustration of the use of a partially 20 duplexed adaptor in the methods of this invention involves ligating such adaptor to a human FR3 region that has been cleaved, as described above, at 5'-ACnGT 3' using HpyCH4III, Bst4CI or TaaI. Table 4 F.2 shows the bottom strand of the 25 double-stranded portion of the adaptor for ligation to the cleaved bottom-strand DNA. Since the HpyCH4III Site is so far to the right (as shown in Table 3), a sequence that includes the AflII-site as well as the XbaI site can be added. This bottom strand portion of 30 the partially-duplexed adaptor, H43.XAExt, incorporates both XbaI and AMlI-sites. The top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments - 38 opposite the XbaI and AflII-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt. H43AExt contains only the AflII-site and is to be used with the top strands H43.ABrl and H43.ABr2 (which have 5 intentional alterations to destroy the AflII-site). After ligation, the desired, captured DNA can be PCR amplified again, if desired, using in the preferred embodiment a primer to the downstream constant region of the antibody gene and a primer to 10 part of the double-standard region of the adapter. The primers may also carry restriction endonuclease sites for use in cloning the amplified DNA. After ligation, and perhaps amplification, of the partially double-stranded adapter to the single 15 stranded amplified DNA, the composite DNA is cleaved at chosen 5' and 3' endonuclease recognition sites. The cleavage sites useful for cloning depend on the phage or phagemid or other vectors into which the cassette will be inserted and the available sites 20 in the antibody genes. Table 19 provides restriction endonuclease data for 75 human light chains. Table 20 shows corresponding data for 79 human heavy chains. In each Table, the endonucleases are ordered by increasing frequency of cutting. In these Tables, Nch is the 25 number of chains cut by the enzyme and Ns is the number of sites (some chains have more than one site). From this analysis, SfiI, NotI, AMlI, ApaLI, and AscI are very suitable. SfiI and NotI are preferably used in pCES1 to insert the heavy-chain 30 display segment. ApaLI and AscI are preferably used in pCES1 to insert the light-chain display segment. BstEII-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/9 (68%) of - 39 heavy-chain genes contain a BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII-Site. An alternative to using BstEII is to cleave via UREs at the end of JH and ligate to a 5 synthetic oligonucleotide that encodes part of CH1. One example of preparing a family of DNA sequences using the methods of this invention involves capturing human CDR 3 diversity. As described above, mRNAs from various autoimmune patients are reverse 10 transcribed into lower strand cDNA. After the top strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide used to cleave the cDNA upstream of CDR3. A partially duplexed synthetic DNA adapter is then annealed to the DNA and the DNA is 15 amplified using a primer to the adapter and a primer to the constant region (after FR4). The DNA is then cleaved using BstEII (in FR4) and a restriction endonuclease appropriate to the partially double stranded adapter (e.g., XbaI and AflII (in FR3)). The 20 DNA is then ligated into a synthetic VH skeleton such as 3-23. One example of preparing a single-stranded DNA that was cleaved using the URE method involves the human Kappa chain. The cleavage site in the sense 25 strand of this chain is depicted in Table 17. The oligonucleotide kapextURE is annealed to the oligonucleotides (kaBROlUR, kaBRO2UR, kaBRO3UR, and kaBRO4UR) to form a partially duplex DNA. This DNA is then ligated to the cleaved soluble kappa chains. The 30 ligation product is then amplified using primers kapextUREPCR and cKForeAsc (which inserts a AscI site after the end of C kappa). This product is then cleaved with ApaLI and AscI and ligated to similarly - 40 cut recipient vector. Another example involves the cleavage of lambda light chains, illustrated in Table 18. After cleavage, an extender (ONLamEx133) and four bridge 5 oligonucleotides (ONLamBl-133, ONLamB2-133, ONLamB3-133, and ONLamB4-133) are annealed to form a partially duplex DNA. That DNA is ligated to the cleaved lambda-chain sense strands. After ligation, the DNA is amplified with ON_Lam133PCR and a forward primer specific to the 10 lambda constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130). In human heavy chains, one can cleave almost all genes in FR4 (downstream, i.e., toward the 3' end of the sense strand, of CDR3) at a BstEII-Site that 15 occurs at a constant position in a very large fraction of human heavy-chain V genes. One then needs a site in FR3, if only CDR3 diversity is to be captured, in FR2,, if CDR2 and CDR3 diversity is wanted, or in FR1, if all the CDR diversity is wanted. These sites are 20 preferably inserted as part of the partially double stranded adaptor. The preferred process of this invention is to provide recipient vectors (e.g., for display and/or expression) having sites that allow cloning of either 25 light or heavy chains. Such vectors are well known and widely used in the art. A preferred phage display vector in accordance with this invention is phage MALIA3. This displays in gene III. The sequence of the phage MALIA3 is shown in Table 21A (annotated) and 30 Table 21B (condensed). The DNA encoding the selected regions of the light or heavy chains can be transferred to the vectors using endonucleases that cut either light or heavy - 41 chains only very rarely. For example, light chains may be captured with ApaLI and AscI. Heavy-chain genes are preferably cloned into a recipient vector having SfiI, NcoI, XbaI, AflII, BstEII, ApaI, and NotI sites. The 5 light chains are preferably moved into the library as ApaLI-AscI fragments. The heavy chains are preferably moved into the library as SfiI-NotI fragments. Most preferably, the display is had on the surface of a derivative of M13 phage. The most 10 preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred 15 vector is stable against rearrangement under the growth conditions used to amplify phage. In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a 20 phagemid vector (e.g., pCES1) that displays and/or expresses the peptide, polypeptide or protein. Such vectors may also be used to store the diversity for subsequent display and/or expression using other vectors or phage. 25 In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a yeast vector. In another embodiment, the mode of display 30 may be through a short linker to anchor domains -- one possible anchor comprising the final portion of M13 III ("IIIstump") and a second possible anchor being the full length III mature protein.

- 42 The IiIstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. III proteins are present the phage is unlikely to delete 5 the antibody genes and phage that do delete these segments receive only a very small growth advantage. For each of the anchor domains, the DNA encodes the w.t. AA sequence, but differs from the w.t. DNA sequence to a very high extent. This will greatly 10 reduce the potential for homologous recombination between the anchor and the w.t. gene that is also present (see Example 6). Most preferably, the present invention uses a complete phage carrying an antibiotic-resistance gene 15 (such as an ampicillin-resistance gene) and the display cassette. Because the w.t. iii and possibly viii genes are present, the w.t. proteins are also present. The display cassette is transcribed from a regulatable promoter (e.g., PLacZ). Use of a regulatable promoter 20 allows control of the ratio of the fusion display gene to the corresponding w.t. coat protein. This ratio determines the average number of copies of the display fusion per phage (or phagemid) particle. Another aspect of the invention is a method 25 of displaying peptides, polypeptides or proteins (and particularly Fabs) on filamentous phage. In the most preferred embodiment this method displays FABs and comprises: a) obtaining a cassette capturing a diversity of 30 segments of DNA encoding the elements: P e::RBS1::SS1::VL::CL::stop::RBS2::SS2::VH::CH1:: linker::anchor::stop::, - 43 where Peg is a regulatable promoter, RBS1 is a first ribosome binding site, SS1 is a signal sequence operable in the host strain, VL is a member of a 5 diverse set of light-chain variable regions, CL is a light-chain constant region, stop is one or more stop codons, RBS2 is a second ribosome binding site, SS2 is a second signal sequence operable in the host strain, VH is a member of a diverse set of heavy-chain variable 10 regions, CHl is an antibody heavy-chain first constant domain, linker is a sequence of amino acids of one to about 50 residues, anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons; and 15 b) positioning that cassette within the phage genome to maximize the viability of the phage and to minimize the potential for deletion of the cassette or parts thereof. 20 The DNA encoding the anchor protein in the above preferred cassette should be designed to encode the same (or a closely related) amino acid sequence as is found in one of the coat proteins of the phage, but with a distinct DNA sequence. This is to prevent 25 unwanted homologous recombination with the w.t. gene. In addition, the cassette should be placed in the intergenic region. The positioning and orientation of the display cassette can influence the behavior of the phage. 30 In one embodiment of the invention, a transcription terminator may be placed after the second stop of the display cassette above (e.g., Trp). This will reduce interaction between the display cassette - 44 and other genes in the phage antibody display vector. In another embodiment of the methods of this invention, the phage or phagemid can display and/or express proteins other than Fab, by replacing the Fab 5 portions indicated above, with other protein genes. Various hosts can be used the display and/or expression aspect of this invention. Such hosts are well known in the art. In the preferred embodiment, where Fabs are being displayed and/or expressed, the 10 preferred host should grow at 30"C and be RecA- (to reduce unwanted genetic recombination) and EndA- (to make recovery of RF DNA easier). It is also preferred that the host strain be easily transformed by electroporation. 15 XL1-Blue MRF' satisfies most of these preferences, but does not grow well at 30 0 C. XLl-Blue MRF' does grow slowly at 38'C and thus is an acceptable host. TG-1 is also an acceptable host although it is RecA* and EndA*. XL1-Blue MRF' is more preferred for 20 the intermediate host used to accumulate diversity prior to final construction of the library. After display and/or expression, the libraries of this invention may be screened using well known and conventionally used techniques. The selected 25 peptides, polypeptides or proteins may then be used to treat disease. Generally, the peptides, polypeptides or proteins for use in therapy or in pharmaceutical compositions are produced by isolating the DNA encoding the desired peptide, polypeptide or protein from the 30 member of the library selected. That DNA is then used in conventional methods to produce the peptide, polypeptides or protein it encodes in appropriate host cells, preferably mammalian host cells, e.g., CHO - 45 cells. After isolation, the peptide, polypeptide or protein is used alone or with pharmaceutically acceptable compositions in therapy to treat disease. EXAMPLES 5 Example 1: RACE amplification of heavy and light chain antibody repertoires from autoimmune patients. Total RNA was isolated from individual blood samples (50 ml) of 11 patients using a RNAzolTM kit (CINNA/Biotecx), as described by the manufacturer. The 10 patients were diagnosed as follows: 1. SLE and phospholipid syndrome 2. limited systemic sclerosis 3. SLE and Sjogren syndrome 4. Limited Systemic sclerosis 15 5. Reumatoid Arthritis with active vasculitis 6. Limited systemic sclerosis and Sjogren Syndrome 7. Reumatoid Artritis and (not active) vasculitis 8. SLE and Sjogren syndrome 9. SLE 20 10. SLE and (active) glomerulonephritis 11. Polyarthritis/ Raynauds Phenomen From these 11 samples of total RNA, Poly-A+ RNA was isolated using Promega PolyATtract@ mRNA Isolation kit (Promega). 25 250 ng of each poly-A+ RNA sample was used to amplify antibody heavy and light chains with the GeneRAacerTM kit (Invitrogen cat no. L1500-01). A schematic overview of the RACE procedure is shown in - 46 FIG. 3. Using the general protocol of the GeneRAacer kit, an RNA adaptor was ligated to the 5'end of all mRNAs. Next, a reverse transcriptase reaction was 5 performed in the presence of oligo(dT15) specific primer under conditions described by the manufacturer in the GeneRAacer m kit. 1/5 of the cDNA from the reverse transcriptase reaction was used in a 20 ul PCR 10 reaction. For amplification of the heavy chain IgM repertoire, a forward primer based on the CH1 chain of IgM [HuCmFOR] and a backward primer based on the ligated synthetic adaptor sequence [5'A] were used. (See Table 22) 15 For amplification of the kappa and lambda light chains, a forward primer that contains the 3' coding-end of the cDNA [HuCkFor and HuCLFor2+HuCLfor7] and a backward primer based on the ligated synthetic adapter sequence [5'A) was used (See Table 22). 20 Specific amplification products after 30 cycles of primary PCR were obtained. FIG. 4 shows the amplification products obtained after the primary PCR reaction from 4 different patient samples. 8 ul primary PCR product 25 from 4 different patients was analyzed on a agarose gel [labeled 1,2, 3 and 4]. For the heavy chain, a product of approximately 950 nt is obtained while for the kappa and lambda light chains the product is approximately 850 nt. M1-2 are molecular weight markers. 30 PCR products were also analyzed by DNA sequencing [10 clones from the lambda, kappa or heavy chain repertoires] . All sequenced antibody genes recovered contained the full coding sequence as well as - 47 the 5' leader sequence and the V gene diversity was the expected diversity (compared to literature data). 50 ng of all samples from all 11 individual amplified samples were mixed for heavy, lambda light or 5 kappa light chains and used in secondary PCR reactions. In all secondary PCRs approximately 1 ng template DNA from the primary PCR mixture was used in multiple 50 ul PCR reactions [25 cycles]. For the heavy chain, a nested biotinylated 10 forward primer [HuCm-Nested] was used, and a nested 5'end backward primer located in the synthetic adapter-sequence [5'NA) was used. The 5'end lower-strand of the heavy chain was biotinylated. For the light chains, a 5'end biotinylated 15 nested primer in the synthetic adapter was used [5'NA] in combination with a 3'end primer in the constant region of Ckappa and Clambda, extended with a sequence coding for the AscI restriction site [ kappa: HuCkForAscI, Lambda: HuCL2-FOR-ASC + HuCL7-FOR-ASC]. 20 [5'end Top strand DNA was biotinylated]. After gel-analysis the secondary PCR products were pooled and purified with Promega Wizzard PCR cleanup. Approximately 25 ug biotinylated heavy chain, lambda and kappa light chain DNA was isolated from the 11 25 patients. Example 2: Capturing kappa chains with BsmAI. A repertoire of human-kappa chain mRNAs was prepared using the RACE method of Example 1 from a 30 collection of patients having various autoimmune diseases.

- 48 This Example followed the protocol of Example 1. Approximately 2 micrograms (ug) of human kappa chain (Igkappa) gene RACE material with biotin attached to 5'-end of upper strand was immobilized as in Example 5 1 on 200 microliters (pL) of Seradyn magnetic beads. The lower strand was removed by washing the DNA with 2 aliquots 200 piL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 seconds for the second aliquot. The beads were neutralized with 200 pL of 10 10 mM Tris (pH 7.5) 100 mM NaCl. The short oligonucleotides shown in Table 23 were added in 40 fold molar excess in 100 pL of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol pH 7.9) to the dry beads. The mixture was incubated at 15 95 0 C for 5 minutes then cooled down to 55*C over 30 minutes. Excess oligonucleotide was washed away with 2 washes of NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 1 mM dithiothreitol pH 7.9). Ten units of BsmAI (NEB) were added in NEB buffer 3 and incubated 20 for 1 h at 55 0 C. The cleaved downstream DNA was collected and purified over a Qiagen PCR purification column (FIGs. 5 and 6). FIG. 5 shows an analysis of digested kappa single-stranded DNA. Approximately 151.5 pmol of 25 adapter was annealed to 3.79 pmol of immobilized kappa single-stranded DNA followed by digestion with 15 U of BsmAI. The supernatant containing the desired DNA was removed and analyzed by 5% polyacrylamide gel along with the remaining beads which contained uncleaved full 30 length kappa DNA. 189 pmol of cleaved single-stranded DNA was purified for further analysis. Five percent of the original full length ssDNA remained on the beads.

- 49 FIG. 6 shows an analysis of the extender cleaved kappa ligation. 180 pmol of pre-annealed bridge/extender was ligated to 1.8 pmol of BsmAI digested single-stranded DNA. The ligated DNA was 5 purified by Qiagen PCR purification column and analyzed on a 5% polyacrylamide gel. Results indicated that the ligation of extender to single-stranded DNA was 95% efficient. A partially double-stranded adaptor was 10 prepared using the oligonucleotide shown in Table 23. The adaptor was added to the single-stranded DNA in 100 fold molar excess along with 1000 units of T4 DNA ligase and incubated overnight at 16 0 C. The excess oligonucleotide was removed with a Qiagen PCR 15 purification column. The ligated material was amplified by PCR using the primers kapPCRtl and kapfor shown in Table 23 for 10 cycles with the program shown in Table 24. The soluble PCR product was run on a gel and 20 showed a band of approximately 700 n, as expected (FIGs. 7 and 8). The DNA was cleaved with enzymes ApaLI and AscI, gel purified, and ligated to similarly cleaved vector pCES1. FIG. 7 shows an analysis of the PCR product 25 from the extender-kappa amplification. Ligated extender-kappa single-stranded DNA was amplified with primers specific to the extender and to the constant region of the light chain. Two different template concentrations, 10 ng versus 50 ng, were used as 30 template and 13 cycles were used to generate approximately 1.5 ug of dsDNA as shown by 0.8% agarose gel analysis.

- 50 FIG. 8 shows an analysis of the purified PCR product from the extender-kappa amplification. Approximately 5 ug of PCR amplified extender-kappa double-stranded DNA was run out on a 0.8% agarose gel, 5 cut out, and extracted with a GFX gel purification column. By gel analysis, 3.5 ug of double-stranded DNA was prepared. The assay for capturing kappa chains with BsmAl was repeated and produced similar results. 10 FIG 9A shows the DNA after it was cleaved and collected and purified over a Qiagen PCR purification column. FIG. 9B shows the partially double-stranded adaptor ligated to the single-stranded DNA. This ligated material was then amplified (FIG. 9C). The gel showed 15 a band of approximately 700 n. Table 25 shows the DNA sequence of a kappa light chain captured by this procedure. Table 26 shows a second sequence captured by this procedure. The closest bridge sequence was complementary to the 20 sequence 5'-agccacc-3', but the sequence captured reads 5'-Tgccacc-3', showing that some mismatch in the overlapped region is tolerated. Example 3: Construction of Synthetic CDRl and CDR2 Diversity in V-3-23 VH Framework. 25 Synthetic diversity in Complementary Determinant Region (CDR) 1 and 2 was created in the 3 23 VH framework in a two step process: first, a vector containing the 3-23 VH framework was constructed; and then, a synthetic CDR 1 and 2 was assembled and cloned 30 into this vector.

- 51 For construction of the 3-23 VH framework, 8 oligonucleotides and two PCR primers (long oligonucleotides - TOPFR1A, BOTFR1B, BOTFR2, BOTFR3, F06, BOTFR4, ON-vgCl, and ON-vgC2 and primers - SFPRMET and 5 BOTPCRPRIM, shown in Table 27) that overlap were designed based on the Genebank sequence of 3-23 VH framework region. The design incorporated at least one useful restriction site in each framework region, as shown in Table 27. In Table 27, the segments that were 10 synthesized are shown as bold, the overlapping regions are underscored, and the PCR priming regions at each end are underscored. A mixture of these 8 oligos was combined at a final concentration of 2.5uM in a 20ul PCR reaction. 15 The PCR mixture contained 200uM dNTPs, 2.5mM MgCl 2 , 0.02U Pfu TurboTm DNA Polymerase, lU Qiagen HotStart Taq DNA Polymerase, and 1X Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94'C for 30s, 55*C for 30s, and 72*C for 30s. 20 The assembled 3-23 VH DNA sequence was then amplified, using 2.5ul of a 10-fold dilution from the initial PCR in 100ul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5mM MgCl 2 , 0.02U Pfu TurboTM DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, 25 1X Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of luM. The PCR program consisted of 23 cycles at 94 0 C for 30s, 55 0 C for 30s, and 72*C for 60s. The 3-23 VH DNA sequence was digested and cloned into pCES1 (phagemid vector) using 30 the SfiI and BstEII restriction endonuclease sites. All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, MA and used as per the manufacturer's instructions.

- 52 Stuffer sequences (shown in Table 28 and Table 29) were introduced into pCES1 to replace CDR1/CDR2 sequences (900 bases between BspEI and XbaI RE sites) and CDR3 sequences (358 bases between AflII 5 and BstEII) prior to cloning the CDR1/CDR2 diversity. This new vector was termed pCES5 and its sequence is given in Table 29. Having stuffers in place of the CDRs avoids the risk that a parental sequence would be over 10 represented in the library. The stuffer sequences are fragments from the penicillase gene of E. coli. The CDR1-2 stuffer contains restriction sites for BglII, Bsu36I, BclI, XcmI, MluI, PvuII, HpaI, and HincII, the underscored sites being unique within the vector pCES5. 15 The stuffer that replaces CDR3 contains the unique restriction endonuclease site RsrII. A schematic representation of the design for CDR1 and CDR2 synthetic diversity is shown FIG. 10. The design was based on the presence of mutations in 20 DP47/3-23 and related germline genes. Diversity was designed to be introduced at the positions within CDR1 and CDR2 indicated by the numbers in FIG. 10. The diversity at each position was chosen to be one of the three following schemes: 1 = ADEFGHIKLMNPQRSTVWY; 2 = 25 YRWVGS; 3 = PS, in which letters encode equimolar mixes of the indicated amino acids. For the construction of the CDR1 and CDR2 diversity, 4 overlapping oligonucleotides (ON-vgC1, ON_Br12, ON_CD2Xba, and ON-vgC2, shown in Table 27 and 30 Table 30) encoding CDR1/2, plus flanking regions, were designed. A mixture of these 4 oligos was combined at a final concentration of 2.5uM in a 40ul PCR reaction. Two of the 4 oligos contained variegated sequences - 53 positioned at the CDR1 and the CDR2. The PCR mixture contained 200uM dNTPs, 2.5U Pwo DNA Polymerase (Roche), and 1X Pwo PCR buffer with 2mM MgSO 4 . The PCR program consisted of 10 cycles at 94'C for 30s, 600C for 30s, 5 and 720C for 60s. This assembled CDR1/2 DNA sequence was amplified, using 2.5ul of the mixture in 100ul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5U Pwo DNA Polymerase, 1X Pwo PCR Buffer with 2mM MgSO 4 and 2 outside primers at a concentration of luM. The PCR 10 program consisted of 10 cycles at 94*C for 30s, 60*C for 30s, and 72*C for 60s. These variegated sequences were digested and cloned into the 3-23 VH framework in place of the CDR1/2 stuffer. We obtained approximately 7 X 10' independent 15 transformants. CDR3 diversity either from donor populations or from synthetic DNA can be cloned into the vector containing synthetic CDR1 and CDR 2 diversity. A schematic representation of this procedure 20 is shown in FIG. 11. A sequence encoding the FR regions of the human V3-23 gene segment and CDR regions with synthetic diversity was made by oligonucleotide assembly and cloning via BspEl and Xbal sites into a vector that complements the FR1 and FR3 regions. Into 25 this library of synthetic VH segments, the complementary VH-CDR3 sequence (top right) was cloned via Xbal an BstEll sites. The resulting cloned CH genes contain a combination of designed synthetic diversity and natural diversity (see FIG. 11).

- 54 Example 4: Cleavage and ligation of the lambda light chains with HinfI. A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 12A and 12B. 5 Approximately 2 ug of biotinylated human Lambda DNA prepared as described in Example 1 was immobilized on 200 ul Seradyn magnetic beads. The lower strand was removed by incubation of the DNA with 200 ul of 0.1 M NaOH (pH=13) for 3 minutes, the supernatant was removed 10 and an additional washing of 30 seconds with 200 ul of 0.1 M NaOH was performed. Supernatant was removed and the beads were neutralized with 200 ul of 10 mM Tris (pH=7.5), 100 mM NaCl. 2 additional washes with 200 ul NEB2 buffer 2, containing 10 mM Tris (pH=7.9), 50 mM 15 NaCl, 10 mM MgCl2 and 1 mM dithiothreitol, were performed. After immobilization, the amount of ssDNA was estimated on a 5% PAGE-UREA gel. About 0.8 ug ssDNA was recovered and incubated in 100 ul NEB2 buffer 2 containing 80 molar 20 fold excess of an equimolar mix of ONLamlaB7, ONLam2aB7, ONLam3lB7 and ONLam3rB7 [each oligo in 20 fold molar excess] (see Table 31). The mixture was incubated at 95* C for 5 minutes and then slowly cooled down to 5 0 * C over a 25 period of 30 minutes. Excess of oligonucleotide was washed away with 2 washes of 200 ul of NEB buffer 2. 4 U/ug of Hinf I was added and incubated for 1 hour at 500 C. Beads were mixed every 10 minutes. After incubation the sample was purified over 30 a Qiagen PCR purification column and was subsequently analysed on a 5% PAGE-urea gel (see FIG. 13A, cleavage was more than 70% efficient).

- 55 A schematic of the ligation of the cleaved light chains is shown in FIG. 12B. A mix of bridge/extender pairs was prepared from the Brg/Ext oligo's listed in Table 31 (total molar excess 100 5 fold) in 1000 U of T4 DNA Ligase (NEB) and incubated overnight at 16' C. After ligation of the DNA, the excess oligonucleotide was removed with a Qiagen PCR purification column and ligation was checked on a Urea-PAGE gel (see FIG. 13B; ligation was more than 95% 10 efficient). Multiple PCRs were performed containing 10 ng of the ligated material in an 50 ul PCR reaction using 25 pMol ON lamPlePCR and 25 pmol of an equimolar mix of Hu-CL2AscI/HuCL7AscI primer (see Example 1). 15 PCR was performed at 60* C for 15 cycles using Pfu polymerase. About 1 ug of dsDNA was recovered per PCR (see FIG. 13C) and cleaved with ApaLl and AscI for cloning the lambda light chains in pCES2. Example 5: Capture of human heavy-chain CDR3 20 population. A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 14A and 14B. Approximately 3 ug of human heavy-chain (IgM) 25 gene RACE material with biotin attached to 5'-end of lower strand was immobilized on 300 uL of Seradyn magnetic beads. The upper strand was removed by washing the DNA with 2 aliquots 300 uL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 30 seconds for the second aliquot. The beads were neutralized with 300 uL of 10 mM Tris (pH 7.5) 100 mM NaCl. The REdaptors (oligonucleotides used to make - 56 single-stranded DNA locally double-stranded) shown in Table 32 were added in 30 fold molar excess in 200 uL of NEB buffer 4 (50 mM Potasium Acetate, 20 mM Tris-Acetate, 10 mM Magnesuim Acetate, 1 mM 5 dithiothreitol pH 7.9) to the dry beads. The REadaptors were incubated with the single-stranded DNA at 80 *C for 5 minutes then cooled down to 55 *C over 30 minutes. Excess REdaptors were washed away with 2 washes of NEB buffer 4. Fifteen units of HpyCH4III 10 (NEB) were added in NEB buffer 4 and incubated for 1 hour at 55 *C. The cleaved downstream DNA remaining on the beads was removed from the beads using a Qiagen Nucleotide removal column (see FIG. 15). The Bridge/Extender pairs shown in Table 33 15 were added in 25 molar excess along with 1200 units of T4 DNA ligase and incubated overnight at 16 "C. Excess Bridge/Extender was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using primers H43.XAExtPCR2 and 20 Hucumnest shown in Table 34 for 10 cycles with the program shown in Table 35. The soluble PCR product was run on a gel and showed a band of approximately 500 n, as expected (see FIG. 15B). The DNA was cleaved with enzymes SfiI and 25 NotI, gel purified, and ligated to similarly cleaved vector PCES1. Example 6: Description of Phage Display Vector CJRAO5, a member of the library built in vector DY3F7. Table 36 contains an annotated DNA sequence 30 of a member of the library, CJRA05, see FIG. 16. Table 36 is to be read as follows: on each line everything - 57 that follows an exclamation mark "!" is a comment. All occurrences of A, C, G, and T before "!" are the DNA sequence. Case is used only to show that certain bases constitute special features, such as restriction sites, 5 ribosome binding sites, and the like, which are labeled below the DNA. CJRA05 is a derivative of phage DY3F7, obtained by cloning an ApaLI to NotI fragment into these sites in DY3F31. DY3F31 is like DY3F7 except that the light chain and heavy chain genes have been 10 replaced by "stuffer" DNA that does not code for any antibody. DY3F7 contains an antibody that binds streptavidin, but did not come from the present library. The phage genes start with gene ii and 15 continue with genes x, v, vii, ix, viii, iii, vi, i, and iv. Gene iii has been slightly modified in that eight codons have been inserted between the signal sequence and the mature protein and the final amino acids of the signal sequence have been altered. This 20 allows restriction enzyme recognition sites EagI and XbaI to be present. Following gene iv is the phage origin of replication (ori). After ori is bla which confers resistance to ampicillin (ApR). The phage genes and bla are transcribed in the same sense. 25 After bla, is the Fab cassette (illustrated in FIG. 17) comprising: a) PlacZ promoter, b) A first Ribosome Binding Site (RBS1), c) The signal sequence form M13 iii, 30 d) An ApaLI RERS, e) A light chain (a kappa L20::JKl shortened by one codon at the V-J boundary in this case), f) An AscI RERS, - 58 g) A second Ribosome Binding Site (RBS2), h) A signal sequence, preferably PelB, which contains, 1) An SfiI RERS, 5 j) A synthetic 3-23 V region with diversity in CDR1 and CDR2, k) A captured CDR3, 1) A partially synthetic J region (FR4 after BstEII), m) CHl, 10 n) A NotI RERS, o) A His6 tag, p) A cMyc tag, q) An amber codon, r) An anchor DNA that encodes the same amino-acid 15 sequence as codons 273 to 424 of M13 iii (as shown in Table 37). s) Two stop codons, t) An AvrII RERS, and u) A trp terminator. 20 The anchor (item r) encodes the same amino-acid sequence as do codons 273 to 424 of M13 iii but the DNA is approximately as different as possible from the wild-type DNA sequence. In Table 36, the III' stump runs from base 8997 to base 9455. Below the 25 DNA, as comments, are the differences with wild-type iii for the comparable codons with "!W.T" at the ends of these lines. Note that Met and Trp have only a single codon and must be left as is. These AA types are rare. Ser codons can be changed at all three base, 30 while Leu and Arg codons can be changed at two. In most cases, one base change can be introduced per codon. This has three advantages: 1) recombination with the wild-type gene carried elsewhere - 59 on the phage is less likely, 2) new restriction sites can be introduced, facilitating construction; and 3) sequencing primers that bind in only one of the two regions can be designed. 5 The fragment of M13 III shown in CJRA05 is the preferred length for the anchor segment. Alternative longer or shorter anchor segments defined by reference to whole mature III protein may also be utilized. 10 The sequence of M13 III consists of the following elements: Signal Sequence::Domain 1 (D1)::Linker 1 (L1)::Domain 2 (D2)::Linker 2 (L2)::Domain 3 (D3)::Transmembrane Segment (TM):: Intracellular anchor (IC) (see Table 38). 15 The pIII anchor (also known as trpIII) preferably consists of D2::L2::D3::TM::IC. Another embodiment for the pIII anchor consists of D2'::L2::D3::TM::IC (where D2' comprises the last 21 residues of D2 with the first 109 residues deleted). A 20 further embodiment of the pIII anchor consists of D2'(C>S)::L2::D3::TM::IC (where D2'(C>S) is D2' with the single C converted to S), and d) D3::TM::IC. Table 38 shows a gene fragment comprising the NotI site, His6 tag, cMyc tag, an amber codon, a 25 recombinant enterokinase cleavage site, and the whole of mature M13 III protein. The DNA used to encode this sequence is intentionally very different from the DNA of wild-type gene iii as shown by the lines denoted "W.T." containing the w.t. bases where these differ 30 from this gene. III is divided into domains denoted "domain 1", "linker 1", "domain 2", "linker 2", "domain 3", "transmembrane segment", and "intracellular anchor".

- 60 Alternative preferred anchor segments (defined by reference to the sequence of Table 38) include: codons 1-29 joined to codons 104-435, deleting 5 domain 1 and retaining linker 1 to the end; codons 1-38 joined to codons 104-435, deleting domain land retaining the rEK cleavage site plus linker 1 to the end from III; codons 1-29 joined to codons 236-435, deleting 10 domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end; codons 1-38 joined to codons 236-435, deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site; 15 codons 1-29 joined to codons 236-435 and changing codon 240 to Ser(e.g., agc), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end; and codons 1-38 joined to codons 236-435 and changing 20 codon 240 to Ser(e.g., agc), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site. The constructs would most readily be made by methods similar to those of Wang and Wilkinson 25 (Biotechniques 2001: 31(4)722-724) in which PCR is used to copy the vector except the part to be deleted and matching restriction sites are introduced or retained at either end of the part to be kept. Table 39 shows the oligonucleotides to be used in deleting parts of 30 the III anchor segment. The DNA shown in Table 38 has an NheI site before the DINDDRMA recombinant enterokinase cleavage site (rEKCS). If NheI is used in the deletion process with this DNA, the rEKCS site - 61 would be lost. This site could be quite useful in cleaving Fabs from the phage and might facilitate capture of very high-afffinity antibodies. One could mutagenize this sequence so that the NheI site would 5 follow the rEKCS site, an Ala Ser amino-acid sequence is already present. Alternatively, one could use SphI for the deletions. This would involve a slight change in amino acid sequence but would be of no consequence. Example 7 : Selection of antigen binders from an 10 enriched library of human antibodies using phage vector DY3F31. In this example the human antibody library used is described in de Haard et al., (Journal of Biological Chemistry, 274 (26): 18218-30 (1999). This 15 library, consisting of a large non-immune human Fab phagemid library, was first enriched on antigen, either on streptavidin or on phenyl-oxazolone (phOx). The methods for this are well known in the art. Two preselected Fab libraries, the first one selected once 20 on immobilized phOx-BSA (Rl-ox) and the second one selected twice on streptavidin (R2-strep), were chosen for recloning. These enriched repertoires of phage antibodies, in which only a very low percentage have 25 binding activity to the antigen used in selection, were confirmed by screening clones in an ELISA for antigen binding. The selected Fab genes were transferred from the phagemid vector of this library to the DY3F31 vector via ApaLl-Notl restriction sites. 30 DNA from the DY3F31 phage vector was pretreated with ATP dependent DNAse to remove - 62 chromosomal DNA and then digested with ApaLl and Notl. An extra digestion with AscI was performed in between to prevent self-ligation of the vector. The ApaLl/NotI Fab fragment from the preselected libraries was 5 subsequently ligated to the vector DNA and transformed into competent XL1-blue MRF' cells. Libraries were made using vector:insert ratios of 1:2 for phOx-library and 1:3 for STREP library, and using 100 ng ligated DNA per 50 pl of 10 electroporation-competent cells (electroporation conditions : one shock of 1700 V, 1 hour recovery of cells in rich SOC medium, plating on amplicillin containing agar plates). This transformation resulted in a library 15 size of 1.6 x 10' for Rl-ox in DY3F31 and 2.1 x 106 for R2-strep in DY3F31. Sixteen colonies from each library were screened for insert, and all showed the correct size insert (±1400 bp) (for both libraries). Phage was prepared from these Fab libraries 20 as follows. A representative sample of the library was inoculated in medium with ampicillin and glucose, and at OD 0.5, the medium exchanged for ampicillin and 1 mM IPTG. After overnight growth at 37 *C, phage was harvested from the supernatant by PEG-NaCl 25 precipitation. Phage was used for selection on antigen. Ri-ox was selected on phOx-BSA coated by passive adsorption onto immunotubes and R2-strep on streptavidin coated paramagnetic beads (Dynal, Norway), in procedures described in de Haard et. al. and Marks 30 et. al., Journal of Molecular Biology, 222(3): 581-97 (1991). Phage titers and enrichments are given in Table 40.

- 63 Clones from these selected libraries, dubbed R2-ox and R3-strep respectively, were screened for binding to their antigens in ELISA. 44 clones from each selection were picked randomly and screened as 5 phage or soluble Fab for binding in ELISA. For the libraries in DY3F31, clones were first grown in 2TY-2% glucose-50 pg/ml AMP to an OD600 of approximately 0.5, and then grown overnight in 2TY-50 ug/ml AMP +/- 1mM IPTG. Induction with IPTG may result in the production 10 of both phage-Fab and soluble Fab. Therefore the (same) clones were also grown without IPTG. Table 41 shows the results of an ELISA screening of the resulting supernatant, either for the detection of phage particles with antigen binding (Anti-M13 HRP = 15 anti-phage antibody), or for the detection of human Fabs, be it on phage or as soluble fragments, either with using the anti-myc antibody 9E10 which detects the myc-tag that every Fab carries at the C-terminal end of the heavy chain followed by a HRP-labeled 20 rabbit-anti-Mouse serum (column 9E10/RAM-HRP), or with anti-light chain reagent followed by a HRP-labeled goat-anti-rabbit antiserum(anti-CK/CL Gar-HRP). The results shows that in both cases antigen-binders are identified in the library, with as 25 Fabs on phage or with the anti-Fab reagents (Table 41). IPTG induction yields an increase in the number of positives. Also it can be seen that for the phOx-clones, the phage ELISA yields more positives than the soluble Fab ELISA, most likely due to the avid 30 binding of phage. Twenty four of the ELISA-positive clones were screened using PCR of the Fab-insert from the vector, followed by digestion with BstNI. This yielded 17 different patterns for the phOx-binding - 64 Fab's in 23 samples that were correctly analyzed, and 6 out of 24 for the streptavidin binding clones. Thus, the data from the selection and screening from this pre-enriched non-immune Fab library show that the 5 DY3F31 vector is suitable for display and selection of Fab fragments, and provides both soluble Fab and Fab on phage for screening experiments after selection. Example 8: Selection of Phage-antibody libraries on streptavidin magnetic beads. 10 The following example describes a selection in which one first depletes a sample of the library of binders to streptavidin and optionally of binders to a non-target (i.e., a molecule other than the target that one does not want the selected Fab to bind). It is 15 hypothesized that one has a molecule, termed a "competitive ligand", which binds the target and that an antibody which binds at the same site would be especially useful. For this procedure Streptavidin Magnetic 20 Beads (Dynal) were blocked once with blocking solution (2% Marvel Milk, PBS (pH 7.4), 0.01% Tween-20 ("2%MPBST")) for 60 minutes at room temperature and then washed five times with 2%MPBST. 450 iL of beads were blocked for each depletion and subsequent 25 selection set. Per selection, 6.25 iL of biotinylated depletion target (1 mg/mL stock in PBST) was added to 0.250 mL of washed, blocked beads (from step 1). The target was allowed to bind overnight, with tumbling, at 30 4 0 C. The next day, the beads are washed 5 times with

PBST.

- 65 Per selection, 0.010 mL of biotinylated target antigen (1 mg/mL stock in PBST) was added to 0.100 mL of blocked and washed beads (from step 1). The antigen was allowed to bind overnight, with 5 tumbling, at 4 0 C. The next day, the beads were washed 5 times with PBST. In round 1, 2 X 1012 up to 10" plaque forming units (pfu) per selection were blocked against non-specific binding by adding to 0.500 mL of 2%MPBS 10 (=2%MPBST without Tween) for 1 hr at RT (tumble). In later rounds, 1011 pfu per selection were blocked as done in round 1. Each phage pool was incubated with 50 pL of depletion target beads (final wash supernatant removed 15 just before use) on a Labquake rotator for 10 min at room temperature. After incubation, the phage supernatant was removed and incubated with another 50 pL of depletion target beads. This was repeated 3 more times using depletion target beads and twice using 20 blocked streptavidin beads for a total of 7 rounds of depletion, so each phage pool required 350 pL of depletion beads. A small sample of each depleted library pool was taken for titering. Each library pool was added to 25 0.100 mL of target beads (final wash supernatant was removed just before use) and allowed to incubate for 2 hours at room temperature (tumble). Beads were then washed as rapidly as possible (e.g.,3 minutes total) with 5 X 0.500 mL PBST and then 30 2X with PBS. Phage still bound to beads after the washing were eluted once with 0.250 mL of competitive ligand (~-l ipM) in PBST for 1 hour at room temperature on a Labquake rotator. The eluate was removed, mixed - 66 with 0.500 mL Minimal A salts solution and saved. For a second selection, 0.500 mL 100 mM TEA was used for elution for 10 min at RT, then neutralized in a mix of 0.250 mL of 1 M Tris, pH 7.4 + 0.500 mL Min A salts. 5 After the first selection elution, the beads can be eluted again with 0.300 mL of non-biotinylated target (1 mg/mL) for 1 hr at RT on a Labquake rotator. Eluted phage are added to 0.450 mL Minimal A salts. Three eluates (competitor from 1st selection, 10 target from 1st selection and neutralized TEA elution from 2nd selection) were kept separate and a small aliquot taken from each for titering. 0.500 mL Minimal A salts were added to the remaining bead aliquots after competitor and target elution and after TEA elution. 15 Take a small aliquot from each was taken for tittering. Each elution and each set of eluted beads was mixed with 2X YT and an aliquot (e.g., 1 mL with 1. E 10/mL) of XL1-Blue MRF' E. coli cells (or other F' cell line) which had been chilled on ice after having been 20 grown to mid-logarithmic phase, starved and concentrated (see procedure below - "Mid-Log prep of XL-1 blue MRF' cells for infection"). After approximately 30 minutes at room temperature, the phage/cell mixtures were spread onto 25 Bio-Assay Dishes (243 X 243 X 18 mm, Nalge Nunc) containing 2XYT, 1mM IPTG agar. The plates were incubated overnight at 30"C. The next day, each amplified phage culture was harvested from its respective plate. The plate was flooded with 35 mL TBS 30 or LB, and cells were scraped from the plate. The resuspended cells were transferred to a centrifuge bottle. An additional 20 mL TBS or LB was used to remove any cells from the plate and pooled with the - 67 cells in the centrifuge bottle. The cells were centrifuged out, and phage in the supernatant was recovered by PEG precipitation. Over the next day, the amplified phage preps were titered. 5 In the first round, two selections yielded five amplified eluates. These amplified eluates were panned for 2-3 more additional rounds of selection using -1. E 12 input phage/round. For each additional round, the depletion and target beads were prepared the 10 night before the round was initiated. For the elution steps in subsequent rounds, all elutions up to the elution step from which the amplified elution came from were done, and the previous elutions were treated as washes. For the 15 bead infection amplified phage, for example, the competitive ligand and target elutions were done and then tossed as washes (see below). Then the beads were used to infect E. coli. Two pools, therefore, yielded a total of 5 final elutions at the end of the 20 selection. 1st selection set A. Ligand amplified elution: elute w/ ligand for 1 hr, keep as elution 25 B. Target amplified elution: elute w/ ligand for 1 hr, toss as wash elute w/ target for 1 hr, keep as elution C. Bead infect. amp. elution: elute w/ ligand for 1 hr, toss as wash elute w/ target 30 for 1 hr, toss as wash elute w/ cell infection, keep as elution - 68 2nd selection set A. TEA amplified elution; elute w/ TEA 10min, keep as elution B. Bead infect. amp. elution; elute w/ 5 TEA 10min, toss as wash elute w/ cell infection, keep as elution Mid-log prep of XL1 blue MRF' cells for infection (based on Barbas et al. Phage Display manual procedure) Culture XL1 blue MRF' in NZCYM (12.5 mg/mL 10 tet) at 37 0 C and 250 rpm overnight. Started a 500 mL culture in 2 liter flask by diluting cells 1/50 in NZCYM/tet (10 mL overnight culture added) and incubated at 37*C at 250 rpm until OD600 of 0.45 (1.5-2 hrs) was reached. Shaking was reduced to 100 rpm for 10 min. 15 When OD600 reached between 0.55-0.65, cells were transferred to 2 x 250 mL centrifuge bottles, centrifuged at 600 g for 15 min at 4*C. Supernatant was poured off. Residual liquid was removed with a pipette. 20 The pellets were gently resuspended (not pipetting up and down) in the original volume of 1 X Minimal A salts at room temp. The resuspended cells were transferred back into 2-liter flask, shaken at 100 rpm for 45 min at 37*C. This process was performed in 25 order to starve the cells and restore pili. The cells were transferred to 2 x 250 mL centrifuge bottles, and centrifuged as earlier. The cells were gently resuspended in ice cold Minimal A salts (5 mL per 500 mL original culture).

- 69 The cells were put on ice for use in infections as soon as possible. The phage eluates were brought up to 7.5 mL with 2XYT medium and 2.5 mL of cells were added. Beads 5 were brought up to 3 mL with 2XYT and 1 mL of cells were added. Incubated at 37oC for 30 min. The cells were plated on 2XYT, 1 mM IPTG agar large NUNC plates and incubated for 18 hr at 30 0 C. Example 9: Incorporation of synthetic region in FR1/3 10 region. Described below are examples for incorporating of fixed residues in antibody sequences for light chain kappa and lambda genes, and for heavy chains. The experimental conditions and 15 oligonucleotides used for the examples below have been described in previous examples (e.g., Examples 3 & 4). The process for incorporating fixed FR1 residues in an antibody lambda sequence consists of 3 steps (see FIG. 18): (1) annealing of single-stranded 20 DNA material encoding VL genes to a partially complementary oligonucleotide mix (indicated with Ext and Bridge), to anneal in this example to the region encoding residues 5-7 of the FRl of the lambda genes (indicated with X..X; within the lambda genes the 25 overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VL gene. In this process the first few residues of all lambda genes will be encoded by the 30 sequences present in the oligonucleotides (Ext., Bridge - 70 or PCRpr). After the PCR, the lambda genes can be cloned using the indicated restriction site for ApaLI. The process for incorporating fixed FR1 residues in an antibody kappa sequence (FIG. 19) 5 consists of 3 steps : (1) annealing of single-stranded DNA material encoding VK genes to a partially complementary oligonucleotide mix (indicated with Ext and Bri), to anneal in this example to the region encoding residues 8-10 of the FRl of the kappa genes 10 (indicated with X..X; within the kappa genes the overlap may sometimes not be perfect) ; (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VK gene. In this process the first few 15 (8) residues of all kappa genes will be encode by the sequences present in the oligonucleotides (Ext., Bridge or PCRpr.). After the PCR, the kappa genes can be cloned using the indicated restriction site for ApaLI. The process of incorporating fixed FR3 20 residues in a antibody heavy chain sequence (FIG. 20) consists of 3 steps : (1) annealing of single-stranded DNA material encoding part of the VH genes (for example encoding FR3, CDR3 and FR4 regions) to a partially complementary oligonucleotide mix (indicated with Ext 25 and Bridge), to anneal in this example to the region encoding residues 92-94 (within the FR3 region) of VH genes (indicated with X..X; within the VH genes the overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the 30 indicated primer ('PCRpr') and for example one primer based within the VH gene (such as in the FR4 region). In this process certain residues of all VH genes will be encoded by the sequences present in the - 71 oligonucleotides used here, in particular from PCRpr (for residues 70-73), or from Ext/Bridge oligonucleotides (residues 74-91). After the PCR, the partial VH genes can be cloned using the indicated 5 restriction site for XbaI. It will be understood that the foregoing is only illustrative of the principles of this invention and that various modifications can be made by those skilled in the art without departing from the scope of 10 and sprit of the invention. The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, 15 unless in the context or usage an exclusive interpretation of the term is required. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

- 72 Table 1: Human GLG FR3 sequences VH1 ! 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 agg gtc acc atg acc agg gac acg tcc atc agc aca gcc tac atg 5 81 82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92 gag ctg agc agg ctg aga tct gac gac acg gcc gtg tat tac tgt ! 93 94 95 gcg aga ga 1-02# 1 aga gtc acc att acc agg gac aca tcc gcg agc aca gcc tac atg 10 gag ctg agc age ctg aga tct gaa gac acg got gtg tat tac tgt gcg aga ga 1-03# 2 aga gtc acc atg acc agg aac acc tcc ata agc aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt gcg aga gg 1-08# 3 15 aga gtc acc atg acc aca gac aca toc acg ago aca gcc tac atg gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgt gcg aga ga 1-18# 4 aga gtc acc atg acc gag gac aca tct aca gac aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 20 gca aca ga 1-24# 5 aga gtc acc att acc agg gac agg tct atg agc aca gcc tac atg gag ctg agc ago ctg aga tct gag gac aca gcc atg tat tac tgt gca aga ta 1-45# 6 aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac atg 25 gag ctg ago ago ctg aga tct gag gac acg goc gtg tat tac tgt gcg aga ga ! 1-46# 7 aga gtc acc att acc agg gac atg tcc aca agc aca gcc tac atg gag ctg agc agc ctg aga tcc gag gac acg gcc gtg tat tac tgt gcg gca ga 1-58# 8 30 aga gto acg att acc gcg gao gaa toc acg ago aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt gcg aga ga 1-69# 9 aga gtc acg att acc gcg gac aaa tcc acg ago aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 35 gcg aga ga 1-e# 10 aga gtc acc ata acc gcg gac acg tct aca gac aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt gca aca ga ! 1-f# 11 - 73 VH2 agg etc acc atc acc aag gac acc tcc aaa aac cag gtg gtc ctt aca atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt gca cac aga c! 2-05# 12 5 agg ctc acc atc tcc aag gac acc tcc aaa agc cag gtg gte ctt acc atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt gca cgg ata c! 2-26# 13 agg ctc acc atc tcc aag gac acc tcc aaa aac cag gtg gtc ctt aca atg acc aac atg gac cct gtg gac aca gcc acg tat tac tgt 10 gca cgg ata c! 2-70# 14 VH3 cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg aga ga 3-07# 15 15 cga tte acc ato tcc aga gac aac gcc aag aac toc ctg tat ctg caa atg aac agt ctg aga gct gag gac acg gcc ttg tat tac tgt gca aaa gat a! 3-09#16 cga ttc acc atc tcc agg gac aac gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt 20 geg aga ga ! 3-11# 17 cga ttc acc atc tcc aga gaa aat gcc aag aac tcc ttg tat ctt caa atg aac agc ctg aga gcc ggg gac acg gct gtg tat tac tgt gca aga ga ! 3-13# 18 aga ttc acc atc tca aga gat gat tca aaa aac acg ctg tat ctg 25 caa atg aac ago ctg aaa ace gag gac aca gcc gtg tat tac tgt acc aca ga ! 3-15# 19 cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa atg aac agt ctg aga gcc gag gac acg gcc ttg tat cac tgt gcg aga ga ! 3-20# 20 30 oga tte acc ate toc aga gao aac gcc aag aac toa ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg aga ga ! 3-21# 21 cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg aac ago ctg aga gcc gag gac acg gcc gta tat tac tgt 35 gcg aaa ga ! 3-23# 22 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg aac agc ctg aga gct gag gac acg got gtg tat tac tgt gcg aaa ga ! 3-30# 23 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg 40 oaa atg aac ago ctg aga got gag gao acg got gtg tat tac tgt gcg aga ga ! 3303# 24 - 74 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt gcg aaa ga ! 3305# 25 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg 5 caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg aga ga ! 3-33# 26 cga ttc acc atc tcc aga gac aac agc aaa aac tcc ctg tat ctg caa atg aac agt ctg aga act gag gac acc gcc ttg tat tac tgt gca aaa gat a! 3-43#27 10 cga ttc acc ate tcc aga gac aat gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga ga ! 3-48# 28 aga ttc acc atc tca aga gat ggt tcc aaa agc atc gcc tat ctg caa atg aac agc ctg aaa acc gag gac aca gcc gtg tat tac tgt 15 act aga ga ! 3-49# 29 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt gcg aga ga ! 3-53# 30 aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt 20 caa atg ggc agc ctg aga gct gag gac atg gct gtg tat tac tgt gcg aga ga 3-64# 31 aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt gcg aga ga ! 3-66# 32 25 aga ttc acc atc tca aga gat gat tca aag aac tca ctg tat ctg caa atg aac agc ctg aaa acc gag gac acg gcc gtg tat tac tgt gct aga ga 3-72# 33 agg ttc acc atc tcc aga gat gat tca aag aac acg gcg tat ctg caa atg aac agc ctg aaa acc gag gac acg gcc gtg tat tac tgt 30 act aga ca 3-73# 34 cga ttc acc atc tcc aga gac aac gcc aag aac acg ctg tat ctg caa atg aac agt ctg aga gcc gag gac acg gct gtg tat tac tgt gca aga ga ! 3-74# 35 aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg cat ctt 35 caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt aag aaa ga 3-d# 36 VH4 cga gtc acc ata tca gta gac aag tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt 40 gcg aga ga ! 4-04# 37 cga gtc acc atg tca gta gac acg tcc aag aac cag ttc tcc ctg - 75 aag ctg agc tct gtg acc gcc gtg gac acg gcc gtg tat tac tgt gcg aga aa 4-28# 38 cga gtt acc ata tca gta gac acg tct aag aac cag ttc tcc ctg aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt 5 gcg aga ga 4301# 39 cga gtc acc ata tca gta gac agg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt gcc aga ga 4302# 40 cga gtt acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 10 aag ctg agc tct gtg act gcc gca gac acg gcc gtg tat tac tgt gcc aga ga 4304# 41 cga gtt acc ata tca qta gac acg tct aag aac cag ttc tcc ctg aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt gcg aga ga 4-31# 42 15 cga gte acc ata tca gta gac acg tec aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gcg gac acg gct gtg tat tac tgt gcg aga ga 4-34# 43 cga gtc acc ata tcc gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg gct gtg tat tac tgt 20 gcg aga ca 4-39# 44 cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt gcg aga ga 4-59# 45 cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 25 aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt gcg aga ga 4-61# 46 cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg gcc gtg tat tac tgt gcg aga ga 4-b# 47 30 ! VHS cag gtc acc atc tca gcc gac aag tcc atc agc acc gcc tac ctg cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt gcg aga ca ! 5-51# 48 cac gtc acc atc tca gct gac aag tcc atc agc act gcc tac ctg 35 cag tgg ago agc ctg aag gcc tcg gac acc gcc atg tat tac tgt gcg aga 5-a# 49 VH6 cga ata acc atc aac cca gac aca tcc aag aac cag ttc tcc ctg cag ctg aac tct gtg act ccc gag gac acg gct gtg tat tac tgt 40 gca aga ga ! 6-1# 50 VH7 - 76 cgg ttt gtc ttc tcc ttg gac acc tct gtc agc acg gca tat ctg cag atc tgc agc cta aag gct gag gac act gcc gtg tat tac tgt gcg aga ga ! 74.1# 51 - 77 Table 2: Enzymes that either cut 15 or more human GLGs or have 5+-base recognition in FR3 Typical entry: REname Recognition #sites GLGid#:base# GLGid#:base# GLGid#:base#..... 5 BstEII Ggtnacc 2 1: 3 48: 3 There are 2 hits at base# 3 10 MaeIII gtnac 36 1: 4 2: 4 3: 4 4: 4 5: 4 6: 4 2: 4 8: 4 9: 4 10: 4 11: 4 37: 4 37: 58 38: 4 38: 58 39: 4 39: 58 40: 4 40: 58 41: 4 41: 58 42: 4 42: 58 43: 4 15 43: 58 44: 4 44: 58 45: 4 45: 58 46: 4 46: 58 47: 4 47: 58 48: 4 49: 4 50: 58 There are 24 hits at base# 4 Tsp45I gtsac 33 20 1: 4 2: 4 3: 4 4: 4 5: 4 6: 4 7: 4 8: 4 9: 4 10: 4 11: 4 37: 4 37: 58 38: 4 38: 58 39: 58 40: 4 40: 58 41: 58 42: 58 43: 4 43: 58 44: 4 44: 58 45: 4 45: 58 46: 4 46: 58 47: 4 47: 58 25 48: 4 49: 4 50: 58 There are 21 hits at base# 4 HphI tcacc 45 1: 5 2: 5 3: 5 4: 5 5: 5 6: 5 30 7: 5 8: 5 11: 5 12: 5 12: 11 13: 5 14: 5 15: 5 16: 5 17: 5 18: 5 19: 5 20: 5 21: 5 22: 5 23: 5 24: 5 25: 5 26: 5 27: 5 28: 5 29: 5 30: 5 31: 5 32: 5 33: 5 34: 5 35: 5 36: 5 37: 5 35 38: 5 40: 5 43: 5 44: 5 45: 5 46: 5 47: 5 48: 5 49: 5 There are 44 hits at base# 5 - 78 NlaIII CATG 26 1: 9 1: 42 2: 42 3: 9 3: 42 4: 9 4: 42 5: 9 5: 42 6: 42 6: 78 7: 9 7: 42 8: 21 8: 42 9: 42 10: 42 11: 42 5 12: 57 13: 48 13: 57 14: 57 31: 72 38: 9 48: 78 49: 78 There are 11 hits at base# 42 There are 1 hits at base# 48 Could cause raggedness. 10 BsaJI Ccnngg 37 1: 14 2: 14 5: 14 6: 14 7: 14 8: 14 8: 65 9: 14 10: 14 11: 14 12: 14 13: 14 14: 14 15: 65 17: 14 17: 65 18: 65 19: 65 20: 65 21: 65 22: 65 26: 65 29: 65 30: 65 15 33: 65 34: 65 35: 65 37: 65 38: 65 39: 65 40: 65 42: 65 43: 65 48: 65 49: 65 50: 65 51: 14 There are 23 hits at base# 65 There are 14 hits at base# 14 20 AluI AGct 42 1: 47 2: 47 3: 47 4: 47 5: 47 6: 47 7: 47 8: 47 9: 47 10: 47 11: 47 16: 63 23: 63 24: 63 25: 63 31: 63 32: 63 36: 63 25 37: 47 37: 52 38: 47 38: 52 39: 47 39: 52 40: 47 40: 52 41: 47 41: 52 42: 47 42: 52 43: 47 43: 52 44: 47 44: 52 45: 47 45: 52 46: 47 46: 52 47: 47 47: 52 49: 15 50: 47 There are 23 hits at base# 47 30 There are 11 hits at base# 52 Only 5 bases from 47 BlpI GCtnagc 21 1: 48 2: 48 3: 48 5: 48 6: 48 7: 48 8: 48 9: 48 10: 48 11: 48 37: 48 38: 48 35 39: 48 40: 48 41: 48 42: 48 43: 48 44: 48 45: 48 46: 48 47: 48 There are 21 hits at base# 48 - 79 MwoI GCNNNNNnngc 19 1: 48 2: 28 19: 36 22: 36 23: 36 24: 36 25: 36 26: 36 35: 36 37: 67 39: 67 40: 67 41: 67 42: 67 43: 67 44: 67 45: 67 46: 67 5 47: 67 There are 10 hits at base# 67 There are 7 hits at base# 36 DdeI Ctnag 71 10 1: 49 1: 58 2: 49 2: 58 3: 49 3: 58 3: 65 4: 49 4: 58 5: 49 5: 58 5: 65 6: 49 6: 58 6: 65 7: 49 7: 58 7: 65 8: 49 8: 58 9: 49 9: 58 9: 65 10: 49 10: 58 10: 65 11: 49 11: 58 11: 65 15: 58 15 16: 58 16: 65 17: 58 18: 58 20: 58 21: 58 22: 58 23: 58 23: 65 24: 58 24: 65 25: 58 25: 65 26: 58 27: 58 27: 65 28: 58 30: 58 31: 58 31: 65 32: 58 32: 65 35: 58 36: 58 36: 65 37: 49 38: 49 39: 26 39: 49 40: 49 20 41: 49 42: 26 42: 49 43: 49 44: 49 45: 49 46: 49 47: 49 48: 12 49: 12 51: 65 There are 29 hits at base# 58 There are 22 hits at base# 49 Only nine base from 58 There are 16 hits at base# 65 Only seven bases from 58 25 BglII Agatct 11 1: 61 2: 61 3: 61 4: 61 5: 61 6: 61 7: 61 9: 61 10: 61 11: 61 51: 47 There are 10 hits at base# 61 30 BstYI Rgatcy 12 1: 61 2: 61 3: 61 4: 61 5: 61 6: 61 7: 61 8: 61 9: 61 10: 61 11: 61 51: 47 There are 11 hits at base# 61 35 - 80 Hpy188I TCNga 17 1: 64 2: 64 3: 64 4: 64 5: 64 6: 64 7: 64 8: 64 9: 64 10: 64 11: 64 16: 57 20: 57 27: 57 35: 57 48: 67 49: 67 5 There are 11 hits at base# 64 There are 4 hits at base# 57 There are 2 hits at base# 67 Could be ragged. MslI CAYNNnnRTG 44 10 1: 72 2: 72 3: 72 4: 72 5: 72 6: 72 7: 72 8: 72 9: 72 10: 72 11: 72 15: 72 17: 72 18: 72 19: 72 21: 72 23: 72 24: 72 25: 72 26: 72 28: 72 29: 72 30: 72 31: 72 32: 72 33: 72 34: 72 35: 72 36: 72 37: 72 15 38: 72 39: 72 40: 72 41: 72 42: 72 43: 72 44: 72 45: 72 46: 72 47: 72 48: 72 49: 72 50: 72 51: 72 There are 44 hits at base# 72 20 BsiEI CGRYcg 23 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74 25 There are 23 hits at base# 74 EaeI Yggccr 23 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 30 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74 There are 23 hits at base# 74 EagI Cggccg 23 35 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74 There are 23 hits at base# 74 - 81 HaeIII GGcc 27 1: 75 3: 75 4: 75 5: 75 7: 75 8: 75 9: 75 10: 75 11: 75 16: 75 17: 75 20: 75 5 22: 75 30: 75 33: 75 34: 75 37: 75 38: 75 39: 75 .40: 75 41: 75 42: 75 45: 75 46: 75 47: 75 48: 63 49: 63 There are 25 hits at base# 75 10 Bst4CI ACNgt 65*C 63 Sites There is a third isoschismer 1: 86 2: 86 3: 86 4: 86 5: 86 6: 86 7: 34 7: 86 8: 86 9: 86 10: 86 11: 86 12: 86 13: 86 14: 86 15: 36 15: 86 16: 53 16: 86 17: 36 17: 86 18: 86 19: 86 20: 53 15 20: 86 21: 36 21: 86 22: 0 22: 86 23: 86 24: 86 25: 86 26: 86 27: 53 27: 86 28: 36 28: 86 29: 86 30: 86 31: 86 32: 86 33: 36 33: 86 34: 86 35: 53 35: 86 36: 86 37: 86 38: 86 39: 86 40: 86 41: 86 42: 86 43: 86 20 44: 86 45: 86 46: 86 47: 86 48: 86 49: 86 50: 86 51: 0 51: 86 There are 51 hits at base# 86 All the other sites are well away HpyCH4III ACNgt 63 25 1: 86 2: 86 3: 86 4: 86 5: 86 6: 86 7: 34 7: 86 8: 86 9: 86 10: 86 11: 86 12: 86 13: 86 14: 86 15: 36 15: 86 16: 53 16: 86 17: 36 17: 86 18: 86 19: 86 20: 53 20: 86 21: 36 21: 86 22: 0 22: 86 23: 86 30 24: 86 25: 86 26: 86 27: 53 27: 86 28: 36 28: 86 29: 86 30: 86 31: 86 32: 86 33: 36 33: 86 34: 86 35: 53 35: 86 36: 86 37: 86 38: 86 39: 86 40: 86 41: 86 42: 86 43: 86 44: 86 45: 86 46: 86 47: 86 48: 86 49: 86 35 50: 86 51: 0 51: 86 There are 51 hits at base# 86 - 82 HinfI Gantc 43 2: 2 3: 2 4: 2 5: 2 6: 2 7: 2 8: 2 9: 2 9: 22 10: 2 11: 2 15: 2 16: 2 17: 2 18: 2 19: 2 19: 22 20: 2 5 21: 2 23: 2 24: 2 25: 2 26: 2 27: 2 28: 2 29: 2 30: 2 31: 2 32: 2 33: 2 33: 22 34: 22 35: 2 36: 2 37: 2 38: 2 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2 50: 60 10 There are 38 hits at base# 2 MlyI GAGTCNNNNNn 18 2: 2 3: 2 4: 2 5: 2 6: 2 7: 2 8: 2 9: 2 10: 2 11: 2 37: 2 38: 2 15 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2 There are 18 hits at base# 2 PleI gagtc 18 2: 2 3: 2 4: 2 5: 2 6: 2 7: 2 20 8: 2 9: 2 10: 2 11: 2 37: 2 38: 2 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2 There are 18 hits at base# 2 Acil Ccgc 24 2: 26 9: 14 10: 14 11: 14 27: 74 37: 62 25 37: 65 38: 62 39: 65 40: 62 40: 65 41: 65 42: 65 43: 62 43: 65 44: 62 44: 65 45: 62 46: 62 47: 62 47: 65 48: 35 48: 74 49: 74 There are 8 hits at base# 62 There are 8 hits at base# 65 30 There are 3 hits at base# 14 There are 3 hits at base# 74 There are 1 hits at base# 26 There are 1 hits at base# 35 -"- Gcgg 11 35 8: 91 9: 16 10: 16 11: 16 37: 67 39: 67 40: 67 42: 67 43: 67 45: 67 46: 67 There are 7 hits at base# 67 There are 3 hits at base# 16 There are 1 hits at base# 91 - 83 BsiHKAI GWGCWc 20 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 5 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 There are 11 hits at base# 51 Bsp12861 GDGCHc 20 10 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 There are 11 hits at base# 51 15 HgiAI GWGCWc 20 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 20 46: 51 47: 51 There are 11 hits at base# 51 BsoFI GCngc 26 2: 53 3: 53 5: 53 6: 53 7: 53 8: 53 25 8: 91 9: 53 10: 53 11: 53 31: 53 36: 36 37: 64 39: 64 40: 64 41: 64 42: 64 43: 64 44: 64 45: 64 46: 64 47: 64 48: 53 49: 53 50: 45 51: 53 There are 13 hits at base# 53 30 There are 10 hits at base# 64 TseI Gcwgc 17 2: 53 3: 53 5: 53 6: 53 7: 53 8: 53 9: 53 10: 53 11: 53 31: 53 36: 36 45: 64 46: 64 48: 53 49: 53 50: 45 51: 53 35 There are 13 hits at base# 53 - 84 MnlI gagg 34 3: 67 3: 95 4: 51 5: 16 5: 67 6: 67 7: 67 8: 67 9: 67 10: 67 11: 67 15: 67 16: 67 17: 67 19: 67 20: 67 21: 67 22: 67 5 23: 67 24: 67 25: 67 26: 67 27: 67 28: 67 29: 67 .30: 67 31: 67 32: 67 33: 67 34: 67 35: 67 36: 67 50: 67 51: 67 There are 31 hits at base# 67 10 HpyCH4V TGca 34 5: 90 6: 90 11: 90 12: 90 13: 90 14: 90 15: 44 16: 44 16: 90 17: 44 18: 90 19: 44 20: 44 21: 44 22: 44 23: 44 24: 44 25: 44 26: 44 27: 44 27: 90 28: 44 29: 44 33: 44 15 34: 44 35: 44 35: 90 36: 38 48: 44 49: 44 50: 44 50: 90 51: 44 51: 52 There are 21 hits at base# 44 There are 1 hits at base# 52 20 AccI GTmkac 13 5-base recognition 7: 37 11: 24 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 43: 16 44: 16 45: 16 46: 16 47: 16 There are 11 hits at base# 16 25 SacII CCGCgg 8 6-base recognition 9: 14 10: 14 11: 14 37: 65 39: 65 40: 65 42: 65 43: 65 There are 5 hits at base# 65 30 There are 3 hits at base# 14 TfiI Gawtc 24 9: 22 15: 2 16: 2 17: 2 18: 2 19: 2 19: 22 20: 2 21: 2 23: 2 24: 2 25: 2 35 26: 2 27: 2 28: 2 29: 2 30: 2 31: 2 32: 2 33: 2 33: 22 34: 22 35: 2 36: 2 There are 20 hits at base# 2 - 85 BsmAl Nnnnnngagac 19 15: 11 16: 11 20: 11 21: 11 22: 11 23: 11 24: 11 25: 11 26: 11 27: 11 28: 11 28: 56 30: 11 31: 11 32: 11 35: 11 36: 11 44: 87 5 48: 87 There are 16 hits at base# 11 BpmI ctccag 19 15: 12 16: 12 17: 12 18: 12 20: 12 21: 12 10 22: 12 23: 12 24: 12 25: 12 26: 12 27: 12 28: 12 30: 12 31: 12 32: 12 34: 12 35: 12 36: 12 There are 19 hits at base# 12 15 XmnI GAANNnnttc 12 37: 30 38: 30 39: 30 40: 30 41: 30 42: 30 43: 30 44: 30 45: 30 46: 30 47: 30 50: 30 There are 12 hits at base# 30 20 BsrI NCcagt 12 37: 32 38: 32 39: 32 40: 32 41: 32 42: 32 43: 32 44: 32 45: 32 46: 32 47: 32 50: 32 There are 12 hits at base# 32 25 BanII GRGCYc 11 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 There are 11 hits at base# 51 30 Ec1136I GAGctc 11 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 There are 11 hits at base# 51 35 SacI GAGCTc 11 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 There are 11 hits at base# 51 - 86 Table 3: Synthetic 3-23 FR3 of human heavy chains showning positions of possible cleavage sites ! Sites engineered into the synthetic gene are shown in upper case DNA ! with the RE name between vertical bars (as in I XbaI I). 5 ! RERSs frequently found in GLGs are shown below the synthetic sequence ! with the name to the right (as in gtn ac=MaeIII(24), indicating that 24 of the 51 GLGs contain the site). 10 |---FR3-- 89 90 (codon # in R F 15 synthetic 3-23) |cgclttcl 6 Allowed DNA |cgnItty| |agri ga ntc = 20 HinfI(38) ga gtc = PleI(18) ga wtc = TfiI(20) 25 gtn ac = MaeIII (24) gts ac = Tsp45I (21) tc acc = 30 HphI(44) -------- FR3------------------------------------------------- ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 T I S R D N S K N T L Y L Q M 35 lactlatclTCTIAGAlgaclaacltctlaaglaatlactictcltaclttglcaglatgI 51 allowediacnlathItcnIcgn gaylaayltcnlaarlaaylacnittrltayIttricariatgI lagylagri |agy) Ictn| Ictnl I galgac = BsmAI(16) ag ct = AluI(23) 40 citcc ag = BpmI(19) g ctn age = BlpI(21) -87 I I g aan nnn ttc = XmnI(12) I XbaI I tg ca = HpyCH4V(21) * --- FR3------------------------------------------------------ >1 5 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 *N S L R A E D T A V Y Y C A K Iaac IagCITTAIAGglgct IgagigaclaCT IGCAIGtcltacltat itgclgct IaaaI 96 !allowedlaayltcnlttrlcgnlgcnlgarlgaylacnlgcnlgtnltayltayltgyigcnlaarI Iagylctnlagri I I 10 1 1 cc nng g = BsaJI (23) ac rigt = Bst4CI (51) I aga tct =BglII(10) Iac ngt =HpyCH4III(51) I Rga tcY = BstYI(I1) Iac ngt = TaaI (51) I Ic ayn nnn rtc = MslI(44) Icg ryc g = BsiEI (23) 15 I I yg gcc r -EaeI(23) Icg gcc g = EagI (23) I IIg gcc = HaeIII(25) I I gag g = MnlI(31) I jAflII I I PstI I - 88 Table 4: REdaptors, Extenders, and Bridges used for Cleavage and Capture of Human Heavy Chains in FR3. A: HpyCH4V Probes of actual human HC genes !HpyCH4V in FR3 of human HC, bases 35-56; only those with TGca site 5 TGca;10, RE recognition:tgca of length 4 is expected at 10 1 6-1 agttctccctgcagctgaactc 2 3-11,3-07,3-21,3-72,3-48 cactgtatctgcaaatgaacag 10 3 3-09,3-43,3-20 ccctgtatctgcaaatgaacag 4 5-51 ccgcctacctgcagtggagcag 5 3-15,3-30,3-30.5,3-30.3,3-74,3-23,3-33 cgctgtatctgcaaatgaacag 6 7-4.1 cggcatatctgcagatctgcag 7 3-73 cggcgtatctgcaaatgaacag 15 8 5-a ctgcctacctgcagtggagcag 9 3-49 tcgcctatctgcaaatgaacag B: HpyCH4V REdaptors, Extenders, and Bridges B.1 REdaptors Cutting HC lower strand: 20 TmKeller for 100 mM NaCl, zero formamide Edapters for cleavage Tyw T K (ONHCFR36-1) 5'-agttctcccTGCAgctgaactc-3' 68.0 64.5 (ONHCFR36-1A) 5'-ttctcccTGCAgctgaactc-3' 62.0 62.5 (ONHCFR36-1B) 5'-ttctcccTGCAgctgaac-3' 56.0 59.9 25 (ONHCFR33-15) 5'-cgctgtatcTGCAaatgaacag-3' 64.0 60.8 (ONHCFR33-15A) 5'-ctgtatcTGCAaatgaacag-3' 56.0 56.3 (ONHCFR33-15B) 5'-ctgtatcTGCAaatgaac-3' 50.0 53.1 (ONHCFR33-11) 5'-cactgtatcTGCAaatgaacag-3' 62.0 58.9 (ONHCFR35-51) 5'-ccgcctaccTGCAgtggagcag-3' 74.0 70.1 30 1 B.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned XbaI... !D323* cgCttcacTaag tcT aqa gac aaC tcT aag aaT acT ctC taC 35 scab........ designed gene 3-23 gene................

- 89 ! HpyCH4V !. AflII... ! Ttg caG atg aac agc TtA agG 5! B.3 Extender and Bridges Extender (bottom strand): (ON_HCHpyExO1) 5'-cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3' 10 ON_HCHpyExO1 is the reverse complement of 5'-cgCttcacTaag tcT aqa gac aaC tcT aag aaT acT ctC taC Ttg -3' Bridges (top strand, 9-base overlap): 15 (ON_HCHpyBrO16-1) 5'-cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC Ttg CAgctgaac-3' {3'-term C is blocked) 3-15 et al. + 3-11 20 (ON_HCHpyBrO23-15) 5'-cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC Ttg CAaatgaac-3' (3'-term C is blocked 5-51 25 (ON_HCHpyBrO45-51) 5'-cgCttcacTaag tcT aqa gac aaC tcT aag aaT acT ctC taC Ttg CAgtggagc-3' {3'-term C is blocked) PCR primer (top strand) 30 (ONHCHpyPCR) 5'-cgCttcacTaag tcT aga gac-3' C: BIpI Probes from human HC GLGs 1 1-58,1-03,1-08,1-69,1-24,1-45,1-46,1-f,1-e 35 acatggaGCTGAGCagcctgag 2 1-02 acatggaGCTGAGCaggctgag - 90 3 1-18 acatggagctgaggagcctgag 4 5-51,5-a acctgcagtggagcagcctgaa 5 5 3-15,3-73,3-49,3-72 atctgcaaatgaacagcctgaa 6 3303,3-33,3-07,3-11,3-30,3-21,3-23,3305,3-48 atctgcaaatgaacagcctgag 7 3-20,3-74,3-09,3-43 10 atctgcaaatgaacagtctgag 8 74.1 atctgcagatctgcagcctaaa 9 3-66,3-13,3-53,3-d atcttcaaatgaacagcctgag 15 10 3-64 atcttcaaatgggcagcctgag 11 4301,4-28,4302,4-04,4304,4-31,4-34,4-39,4-59,4-61,4-b ccctgaaGCTGAGCtctgtgac 12 6-1 20 ccctgcagctgaactctgtgac 13 2-70,2-05 tccttacaatgaccaacatgga 14 2-26 tccttaccatgaccaacatgga 25 D: BIpI REdaptors, Extenders, and Bridges D.1 REdaptors T." T.K (BlpF3HC1-58) 5'-ac atg gaG CTG AGC agc ctg ag-3' 70 66. 4 30 (BlpF3HC6-1) 5'-cc ctg aag ctg agc tct gtg ac-3' 70 66. !BpF3HC6-1 matches 4-30.1, not 6-1. D.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned 35 BlpI XbaI...

- 91 !D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT ctC taC Ttg caG atg aac AflII... 5 agC TTA AGG D.3 Extender and Bridges Bridges (BlpF3Brl) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg ajGC agc ctg-3' 10 (BlpF3Br2) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg algc tct gtg-3' I lower strand is cut here Extender (BlpF3Ext) 5' -TcAgcTgcAAgTAcAAAgTArTrAcTgTTATcTcTAqA cTgAgTgAAgcg 15 3' BlpF3Ext is the reverse complement of: 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3' 20 (BlpF3PCR) 5'-cgCttcacTcag tcT aga gaT aaC-3' E: HpyCH4III Distinct GLG sequences surrounding site, bases 77-98 1 1 02 #1,118#4,146#7,169#9,le#10,31117,353030,404#37,4301 ccgtgtattactgtgcgagaga 2 103#2,307#15,321#21,3303#24,333#26,348#28,364031,366#32 25 ctgtgtattactgtgcgagaga 3 108#3 ccgtgtattactgtgcgagagg 4 124#5,1f#11 ccgtgtattactgtgcaacaga 30 5 145#6 ccatgtattactgtgcaagata 6 158#8 ccgtgtattactgtgcggcaga 7 205#12 35 ccacatattactgtgcacacag 8 226#13 ccacatattactgtgcacggat - 92 9 270#14 ccacgtattaCtgtgcacggat 10 309#16,343#27 ccttgtattactgtgcaaaaga 5 11 313#18,374035,61#50 ctgtgtattactgtgcaagaga 12 315*19 ccgtgtattactgtaccacaga 13 320#20 10 ccttgtatcactgtgcgagaga 14 323022 ccgtatattactgtgcgaaaga 15 330#23,3305#25 ctgtgtattactgtgcgaaaga 15 16 349#29 ccgtgtattactgtactagaga 17 372#33 ccgtgtattactgtgctagaga 18 373#34 20 ccgtgtattactgtactagaca 19 3d#36 ctgtgtattactgtaagaaaga 20 428#38 ccgtgtattactgtgcgagaaa 25 21 4302#40,4304#41 ccgtgtattactgtgccagaga 22 439144 ctgtgtattacttgcgagaca 23 551048 30 ccatgtattactgtgcgagaca 24 5a#49 ccatgtattactgtgcgaga F: HpyCH4III REdaptors, Extenders, and Bridges F.1 REdaptors 35 ONs for cleavage of HC(lower) in FR3(bases 77-97) For cleavage with HpyCH4III, Bst4CI, or TaaI cleavage is in lower chain before base 88. 77 788 888 888 889 999 999 9 78 901 234 567 890 123 456 7 Tw 40 T. (H43.77.97.1-02#1) 5'-cc gtg tat tAC TGT gcg aga g-3' 6462.6 (H43.77.97.1-03#2) 5'-cp gtg tat tAC TGT gcg aga g-3' 6260.6 (H43.77.97.108#3) 5'-cc gtg tat tAC TGT gcg aga g-3' 6462.6 (H43.77.97.323#22) 5'-cc gti tat tac tgt gcg a*a g-3' 6058.7 45 (H43.77.97.330#23) 5'-ct gtg tat tac tgt gcg a'aa g-3' 6058.7 - 93 (H43.77.97.439#44) 5'-cZ 19tg tat tac tgt gcg aga Z--3' 6260.6 (H43.77.97.551#48) 5 1-cc Atg tat tac tgt gcg aga E-3' 6260.6 (H43.77.97.5a#4'9) 5'-cc a.tg tat tAG TGT gcg aga 1-3' 5858.3 F.2 Extender arnd Bridges 5 XbaI and AlIl sites in bridges are bunged (H43.XABr1) 5'-ggtgtagtga ITCTIA~tlgaclaacI tctlaaglaatl actlctcl tac ttglcaglatgl IaaclactClTTtIAGalactl gagigacl aCT IGCAI Gtc Itac Itat tgt gcg aga-3' (H43.XABr2) 5'-ggtgtagtga 10 ITCTIAGtgaclaacI tctlaaglaatlactlctcl taclttglcaglatgl IaacIacyCITTtIA SIgctIqaqgqacI aCTIGCAI Gtc I tacit tat tgt gcg aaa-3' (H43.XAExt) 5'-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc TgcAAqTAgA gAgTATTcTT AgAgTTgTcT cTAgATcAcT AcAcc-3' 15 !H43.XAExt is the reverse complement of 5'-gqtqtagtqa ITCTIAGAIgaclaacitctlaaglaatlactlctcl taclttglcaglatgl laaclacCITTAIAGgjictipacpigaclaCTIGCAIGtcI tacltat -3' (H43.XAPCR) 5'-ggtqtagtqa ITCTIACA~gaclaac-3' 20 ! XbaI and AflII sites in bridges are bunged (H43.ABrl) 5'-ggtgtagtga laaclactCITTtIAGQicictalqIaclaCTIGCAILtcI tacLttttggaa3 (H43.ABr2) 5'-ggtgtagjtga laaclaqCITTtIAG IgctIqaqIqacI aCTI GCAIGtcl tacl tat tgt gcg aaa-3' 25 (H43 .AExt) 5' -ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3 (H43.AExt) is the reverse complement of 5'-ggtgtagtga laaclapCITTAIA~crlgctigaQigadlaCTI GCAI Gtc Itacltat -3' (H43.APCR) 5'-ggtgtagtga JaacIacrCI TTAIACqIqqtIa-3' 94 J0 0 0 0 0 0 0 u 0 U 0 0 0 0 0 U- 4.)4 - U- 0 4J 4- 41 v' 'UbN ( (.) 0 m 0 4-) CU CL t U u 0 U 0'U 0' 1 o I I 14 ~) 0 m. r-~ (1) 0r M 0 0 0 00r C C CU M U 00 4 0) " to 0 U lt 0 0 in 0 0' W0 D 0 c)0 0' !- D 0C 0..0 0 (n 0 0 0 0 -4 C) m a -4 0 E - 4 0 - 0 A .- D m n N 0 U -) 4 > 4 -4 4 I- 00 u m m '-4~ ~~~ '.0 4J A ' C8 L W C (n 0 m 'l ( - >-4 m 4J 4 Z1 -0 01 m' 0 -4 ~ ~ 'a m 0 N OD . v4 0 0 0 N ( V -4 v4 v- n O l 0 4 k LO 40' %D r4- t 14 (0 4 0 m *n U C -4 (r) D 0 -1 0 D - 0 1 - 41 .1 a, C4 Nmu m m U) 4- NO U0 C r 0 04 1 f fu *0 r- mY 0 1 -4 0 0 '- 4-4 D u 0 . 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'4 '41 41 43 '4 ". 43 V 4.3 ' '4 '4 ". '4 '4 43 43 4. 0A 0 0% a, 0 0, mV m CP 'V m a, 0 0 mV m a, ON 'V 0 0r a A-) 4.3 A.3 W -&J '4 U U '4 4J '4 '4 '4 43 4 A.) 4 '4 '4 '4 '4) 43 4.3 43 U0 V ' U ' U ' 0' U U 0 0U U ' U' U' 0 01 U' U' U - UU U' 0V 0 U 0'U 00 U' 0 0' 'V 'V 0 U U U '4 00 U' u U' u' U U' 00 0 Co 4.3 -W %D0 0 0 0' 0 0l 04 . r) 0 M '4. OD 4,r 0 O 1- -4 .. i (N (C4 D i4 C-4 In ((n qw qD a% ('4 (nl0 L CD Ul 00 0 %.D C- ff LA 0 m' 0 C4 (n UT OD 0 4 H- :0 o 0 0 (N4 L I 0 (Ni r- 0 -4 - i (N ('4 w' r~C- rl- .0 (i f (n4 I to -4 4 .4 -4 4 C14 (N (N) mN Cl Ml Cl Cl ( ) Cl) ml IT -W v L in -o o% a 4 D (N oD %-4 ko 4 (N -Lo ml o 4m 0" -4 -1 0 '-i %0 (n im 04 r) %D NN (n .- 0 .44 %D m C-' t 0 CD 0 v-4 "4 14 C)- 0 0 0 0 0> 0 0) 0 0 0 0 C0 (N 0 0 0 0 0 r- 0 m 0 4 -4 -4 0 0 0D 0 '-1 (N 0 .4 0 > CD 0 0 0 0 00 0 w0 (' 0 -4 .- ~4 -4 0 0> 0> 0 (N '4- 0 0) 0 0 0 .4 H4(' rl m4 v- ' 4 - -4 4 N 0 -4 ml -4 0> v v ml -4 0D a m -4 m4 0 1 .4 S0 V v C.4 m- 0 N 0 (N ml %D 0 0 "4 0 0 0> 0 IA (n 0 H4 0 -4 ml .44 (4 ml m w0 (4 0 0(N 0 -4 IA w0( 0 (N mh m 0 0D 0 0 NN1 I ' .4 W0 (4 I'4V mN 0' wA 40 N4 .I-'. 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- 101 Table 5D: Analysis repeated using only 8 best REdaptors Id Ntot 0 1 2 3 4 5 6 7 8+ 5 1 301 78 101 54 32 16 9 10 1 0 281 102#1 ccgtgtattactgtgcgagaga 2 493 69 155 125 73 37 14 11 3 6 459 103#2 ctgtgtattactgtgcgagaga 3 189 52 45 38 23 18 5 4 1 3 176 108#3 10 ccgtgtattactgtgcgagagg 4 127 29 23 28 24 10 6 5 2 0 114 323#22 ccgtatattactgtgcgaaaga 5 78 21 25 14 11 1 4 2 0 0 72 330#23 ctgtgtattactgtgcgaaaga 6 79 15 17 25 8 11 1 2 0 0 76 15 439#44 ctgtgtattactgtgcgagaca 7 43 14 15 5 5 3 0 1 0 0 42 551#48 ccatgtattactgtgcgagaca 8 307 26 63 72 51 38 24 14 13 6 250 5a#49 ccatgtattactgtgcgaga 20 1 102#1 ccgtgtattactgtgcgagaga ccgtgtattactgtgcgagaga 2 103#2 ctgtgtattactgtgcgagaga .t.................... 3 108#3 ccgtgtattactgtgcgagagg ..................... g 4 323#22 ccgtatattactgtgcgaaaga ....a.............a... 5 330#23 ctgtgtattactgtgcgaaaga .t................a... 25 6 439#44 ctgtgtattactgtgcgagaca .t...................c. 7 551#48 ccatgtattactgtgcgagaca ..a..................c. 8 Sa#49 ccatgtattactgtgcgagaAA .. a ................. AA Seqs with the expected RE site only.......1463 / 1617 Seqs with only an unexpected site......... 0 30 Seqs with both expected and unexpected.... 7 Seqs with no sites.......................... 0 - 102 Table 6: Human HC GLG FRI Sequences VH Exon - Nucleotide sequence alignment VH1 1-02 CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC 5 TCC TGC AAG GCT TCT GGA TAC ACC TTC ACC 1-03 cag gtC cag ctT gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag gtT tcc tgc aag gct tct gga tac acc ttc acT 1-08 cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag gtc tcc tgc aag gct tct gga tac acc ttc acc 10 1-18 cag gtT cag ctg gtg cag tct ggA gct gag gtg aag aag cet ggg gcc tca gtg aag gtc tcc tgc aag gct tct ggT tac acc ttT acc 1-24 cag gtC cag ctg gtA cag tct ggg get gag gtg aag aag cct ggg gcc tca gtg aag gtc tcc tgc aag gTt tcC gga tac acc Ctc acT 1-45 cag Atg cag ctg gtg cag tct ggg gct gag gtg aag aag Act ggg Tcc tca gtg aag gtT 15 tcc tgc aag gct tcC gga tac acc etc acc 1-46 cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag gtT tcc tgc aag gcA tct gga tac acc ttc acc 1-58 caA Atg cag ctg gtg cag tct ggg Cct gag gtg aag aag cct ggg Acc tea gtg aag gtc tcc tgc aag gct tct gga tTc acc ttT acT 20 1-69 cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag gtc tcc tgc aag gct tct gga GGc acc ttc aGc 1-e cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag gtc tcc tgc aag gct tct gga GGc acc ttc aGc 1-f Gag gtC cag ctg gtA cag tct ggg gct gag gtg aag aag cct ggg gcT Aca gtg aaA Atc 25 tcc tgc aag gTt tct gga tac acc ttc acc VH2 2-05 CAG ATC ACC TTG AAG GAG TCT GGT CCT ACG CTG GTG AAA CCC ACA CAG ACC CTC ACG CTG ACC TGC ACC TTC TCT GGG TTC TCA CTC AGC 2-26 cag Gtc acc ttg aag gag tct ggt cct GTg ctg gtg aaa ccc aca Gag acc ctc acg ctg 30 acc tgc acc Gtc tct ggg ttc tca ctc agc 2-70 cag Gtc acc ttg aag gag tct ggt cct Gcg ctg gtg aaa ccc aca cag acc ctc acA ctg acc tgc acc ttc tct ggg ttc tca ctc agc VH3 3-07 GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTG GTC CAG CCT GGG GGG TCC CTG AGA CTC 35 TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT 3-09 gaA gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggC Agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt GAt 3-11 Cag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc Aag cct ggA ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 40 3-13 gag gtg cag ctg gtg gag tot ggg gga gge ttg gtA cag cct ggg ggg tce ctg aga etc tcc tgt gca gcc tct gga ttc acc ttC agt 3-15 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA Aag cct ggg ggg tcc ctT aga ctc tcc tgt gca gcc tct gga ttc acT ttC agt - 103 3-20 gag gtg cag ctg gtg gag tct ggg gga ggT Gtg gtA cGg cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt GAt 3-21 gag gtg cag ctg gtg gag tct ggg gga ggc Ctg gtc Aag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 5 3-23 gag gtg cag ctg Ttg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt agC 3-30 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-30.3 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga ctc 10 tcc tgt gca gcc tct gga tic acc ttC agt 3-30.5 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-33 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga ctc tcc tgt gca gcG tct gga ttc acc ttC agt 15 3-43 gaA gtg cag ctg gtg gag tct ggg gga gTc Gtg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt GAt 3-48 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-49 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag ccA ggg Cgg tcc ctg aga ctc 20 tcc tgt Aca gcT tct gga ttc acc ttt Ggt 3-53 gag gtg cag ctg gtg gag Act ggA gga ggc ttg Atc cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct ggG ttc acc GtC agt 3-64 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 25 3-66 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gic cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc GtC agt 3-72 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggA ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-73 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aAa ctc 30 tcc tgt gca gcc tct ggG ttc acc ttC agt 3-74 gag gtg cag ctg gtg gag tcC ggg gga ggc ttA gtT cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-d gag gtg cag ctg gtg gag tct Cgg gga gTc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc GtC agt 35 VH4 4-04 CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG GGG ACC CTG TCC CTC ACC TGC GCT GTC TCT GGT GGC TCC ATC AGC 4-28 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAC acc ctg tcc ctc acc tgc gct gtc tct ggt TAc tcc atc agc 40 4-30.1 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc atc agc 4-30.2 cag Ctg cag ctg cag gag tcC ggc Tca gga ctg gtg aag cct tcA CAg acc ctg tcc ctc acc tgc gct gtc tct ggt ggc tcc atc agc - 104 4-30.4 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc ct( acc tgc Act gtc tct ggt ggc tcc atc agc 4-31 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc atc agc 5 4-34 cag gtg cag ctA cag Cag tGg ggc Gca gga ctg Ttg aag cct tcg gAg acc ctg tcc ctc acc tgc gct gtc tAt ggt ggG tcc Ttc agT 4-39 cag Ctg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc atc agc 4-59 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc 10 acc tge Act gtc tct ggt gge tec ate agT 4-61 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc Gtc agc 4-b cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc acc tgc get gtc tct ggt TAc tcc atc agc 15 VHs 5-51 GAG GTG CAG CTG GTG CAG TCT GGA GCA GAG GTG AAA AAG CCC GGG GAG TCT CTG AAG ATC TCC TGT AAG GGT TCT GGA TAC AGC TTT ACC 5-a gaA gtg cag ctg gtg cag tct gga gca gag gtg aaa aag ccc qgg gag tct ctg aGg atc tcc tgt aag ggt tct gga tac agc ttt acc 20 VH6 6-1 CAG GTA CAG CTG CAG CAG TCA GGT CCA GGA CTG GTG AAG CCC TCG CAG ACC CTC TCA CTC ACC TGT GCC ATC TCC GGG GAC AGT GTC TCT VH7 7-4.1 CAG GTG CAG CTG GTG CAA TCT GGG TCT GAG TTG AAG AAG CGT GGG GCC TCA GTG AAG GTI 25 TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT - 105 Table 7: RERS sites in Human HC GLG FRIs where there are at least 20 GLGs cut BsgI GTGCAG 71 (cuts 16/14 bases to right) 1: 4 1: 13 2: 13 3: 4 3: 13 4: 13 6: 13 7: 4 7: 13 8: 13 9: 4 9: 13 5 10: 4 10: 13 15: 4 15: 65 16: 4 16: 65 17: 4 17: 65 18: 4 18: 65 19: 4 19: 65 20: 4 20: 65 21: 4 21: 65 22: 4 22: 65 23: 4 23: 65 24: 4 24: 65 25: 4 25: 65 26: 4 26: 65 27: 4 27: 65 28: 4 28: 65 10 29: 4 30: 4 30: 65 31: 4 31: 65 32: 4 32: 65 33: 4 33: 65 34: 4 34: 65 35: 4 35: 65 36: 4 36: 65 37: 4 38: 4 39: 4 41: 4 42: 4 43: 4 45: 4 46: 4 47: 4 48: 4 48: 13 49: 4 49: 13 51: 4 15 There are 39 hits at base# 4 There are 21 hits at base# 65 -"- ctgcac 9 12: 63 13: 63 14: 63 39: 63 41: 63 42: 63 20 44: 63 45: 63 46: 63 BbvI GCAGC 65 1: 6 3: 6 6: 6 7: 6 8: 6 9: 6 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 25 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 30 36: 6 36: 67 37: 6 38: 6 39: 6 40: 6 41: 6 42: 6 43: 6 44: 6 45: 6 46: 6 47: 6 48: 6 49: 6 50: 12 51: 6 There are 43 hits at base# 6 Bolded sites very near sites listed below 35 There are 21 hits at base# 67 -"- gctgc 13 37: 9 38: 9 39: 9 40: 3 40: 9 41: 9 42: 9 44: 3 44: 9 45: 9 46: 9 47: 9 - 106 50: 9 There are 11 hits at base# 9 BsoFI GCngc 78 5 1: 6 3: 6 6: 6 7: 6 8: 6 9: 6 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 10 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 36: 6 36: 67 37: 6 37: 9 38: 6 38: 9 39: 6 39: 9 40: 3 40: 6 40: 9 41: 6 15 41: 9 42: 6 42: 9 43: 6 44: 3 44: 6 44: 9 45: 6 45: 9 46: 6 46: 9 47: 6 47: 9 48: 6 49: 6 50: 9 50: 12 51: 6 There are 43 hits at base# 6 These often occur together. There are 11 hits at base# 9 20 There are 2 hits at base 3 There are 21 hits at base# 67 TseI Gcwgc 78 1: 6 3: 6 6: 6 7: 6 8: 6 9: 6 25 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 30 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 36: 6 36: 67 37: 6 37: 9 38: 6 38: 9 39: 6 39: 9 40: 3 40: 6 40: 9 41: 6 41: 9 42: 6 42: 9 43: 6 44: 3 44: 6 35 44: 9 45: 6 45: 9 46: 6 46: 9 47: 6 47: 9 48: 6 49: 6 50: 9 50: 12 51: 6 There are 43 hits at base# 6 Often together. There are 11 hits at base# 9 - 107 There are 2 hits at base# 3 There are 1 hits at base# 12 There are 21 hits at base# 67 5 MspAlI CMGckg 48 1: 7 3: 7 4: 7 5: 7 6: 7 7: 7 8: 7 9: 7 10: 7 11: 7 15: 7 16: 7 17: 7 18: 7 19: 7 20: 7 21: 7 22: 7 23: 7 24: 7 25: 7 26: 7 27: 7 28: 7 10 29: 7 30: 7 31: 7 32: 7 33: 7 34: 7 35: 7 36: 7 37: 7 38: 7 39: 7 40: 1 40: 7 41: 7 42: 7 44: 1 44: 7 45: 7 46: 7 47: 7 48: 7 49: 7 50: 7 51: 7 There are 46 hits at base# 7 15 PvuII CAGctg 48 1: 7 3: 7 4: 7 5: 7 6: 7 7: 7 8: 7 9: 7 10: 7 11: 7 15: 7 16: 7 17: 7 18: 7 19: 7 20: 7 21: 7 22: 7 20 23: 7 24: 7 25: 7 26: 7 27: 7 28: 7 29: 7 30: 7 31: 7 32: 7 33: 7 34: 7 35: 7 36: 7 37: 7 38: 7 39: 7 40: 1 40: 7 41: 7 42: 7 44: 1 44: 7 45: 7 46: 7 47: 7 48: 7 49: 7 50: 7 51: 7 25 There are 46 hits at base# 7 There are 2 hits at base# 1 Alul AGct 54 1: 8 2: 8 3: 8 4: 8 4: 24 5: 8 30 6: 8 7: 8 8: 8 9: 8 10: 8 11: 8 15: 8 16: 8 17: 8 18: 8 19: 8 20: 8 21: 8 22: 8 23: 8 24: 8 25: 8 26: 8 27: 8 28: 8 29: 8 29: 69 30: 8 31: 8 32: 8 33: 8 34: 8 35: 8 36: 8 37: 8 35 38: 8 39: 8 40: 2 40: 8 41: 8 42: 8 43: 8 44: 2 44: 8 45: 8 46: 8 47: 8 48: 8 48: 82 49: 8 49: 82 50: 8 51: 8 - 108 There are 48 hits at base# 8 There are 2 hits at base# 2 DdeI Ctnag 48 5 1:-26 1: 48 2: 26 2: 48 3: 26 3: 48 4: 26 4: 48 5: 26 5: 48 6: 26 6: 48 7: 26 7: 48 8: 26 8: 48 9: 26 10: 26 11: 26 12: 85 13: 85 14: 85 15: 52 16: 52 17: 52 18: 52 19: 52 20: 52 21: 52 22: 52 10 23: 52 24: 52 25: 52 26: 52 27: 52 28: 52 29: 52 30: 52 31: 52 32: 52 33: 52 35: 30 35: 52 36: 52 40: 24 49: 52 51: 26 51: 48 There are 22 hits at base# 52 52 and 48 never together. There are 9 hits at base# 48 15 There are 12 hits at base# 26 26 and 24 never together. HphI tcacc 42 1: 86 3: 86 6: 86 7: 86 8: 80 11: 86 12: 5 13: 5 14: 5 15: 80 16: 80 17: 80 20 18: 80 20: 80 21: 80 22: 80 23: 80 24: 80 25: 80 26: 80 27: 80 28: 80 29: 80 30: 80 31: 80 32: 80 33: 80 34: 80 35: 80 36: 80 37: 59 38: 59 39: 59 40: 59 41: 59 42: 59 43: 59 44: 59 45: 59 46: 59 47: 59 50: 59 25 There are 22 hits at base# 80 80 and 86 never together There are 5 hits at base# 86 There are 12 hits at base# 59 BssKI Nccngg 50 30 1: 39 2: 39 3: 39 4: 39 5: 39 7: 39 8: 39 9: 39 10: 39 11: 39 15: 39 16: 39 17: 39 18: 39 19: 39 20: 39 21: 29 21: 39 22: 39 23: 39 24: 39 25: 39 26: 39 27: 39 28: 39 29: 39 30: 39 31: 39 32: 39 33: 39 35 34: 39 35: 19 35: 39 36: 39 37: 24 38: 24 39: 24 41: 24 42: 24 44: 24 45: 24 46: 24 47: 24 48: 39 48: 40 49: 39 49: 40 50: 24 50: 73 51: 39 There are 35 hits at base# 39 39 and 40 together twice.

- 109 There are 2 hits at base# 40 BsaJI Ccnngg 47 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 5 8: 40 9: 40 9: 47 10: 40 10: 47 11: 40 15: 40 18: 40 19: 40 20: 40 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 28: 40 29: 40 30: 40 31: 40 32: 40 34: 40 35: 20 35: 40 36: 40 37: 24 38: 24 39: 24 41: 24 10 42: 24 44: 24 45: 24 46: 24 47: 24 48: 40 48: 41 49: 40 49: 41 50: 74 51: 40 There are 32 hits at base# 40 40 and 41 together twice There are 2 hits at base# 41 There are 9 hits at base# 24 15 There are 2 hits at base# 47 BstNI CCwgg 44 PspGI ccwgg ScrFI($M.HpaII) CCwgg 20 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 8: 40 9: 40 10: 40 11: 40 15: 40 16: 40 17: 40 18: 40 19: 40 20: 40 21: 30 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 28: 40 29: 40 30: 40 31: 40 32: 40 33: 40 25 34: 40 35: 40 36: 40 37: 25 38: 25 39: 25 41: 25 42: 25 44: 25 45: 25 46: 25 47: 25 50: 25 51: 40 There are 33 hits at base# 40 30 ScrFI CCngg 50 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 8: 40 9: 40 10: 40 11: 40 15: 40 16: 40 17: 40 18: 40 19: 40 20: 40 21: 30 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 35 28: 40 29: 40 30: 40 31: 40 32: 40 33: 40 34: 40 35: 20 35: 40 36: 40 37: 25 38: 25 39: 25 41: 25 42: 25 44: 25 45: 25 46: 25 - 110 47: 25 48: 40 48: 41 49: 40 49: 41 50: 25 50: 74 51: 40 There are 35 hits at base# 40 There are 2 hits at base# 41 5 EcoO109I RGgnccy 34 1: 43 2: 43 3: 43 4: 43 5: 43 6: 43 7: 43 8: 43 9: 43 10: 43 15: 46 16: 46 17: 46 18: 46 19: 46 20: 46 21: 46 22: 46 10 23: 46 24: 46 25: 46 26: 46 27: 46 28: 46 30: 46 31: 46 32: 46 33: 46 34: 46 35: 46 36: 46 37: 46 43: 79 51: 43 There are 22 hits at base# 46 46 and 43 never together There are 11 hits at base# 43 15 NlaIV GGNncc 71 1: 43 2: 43 3: 43 4: 43 5: 43 6: 43 7: 43 8: 43 9: 43 9: 79 10: 43 10: 79 15: 46 15: 47 16: 47 17: 46 17: 47 18: 46 18: 47 19: 46 19: 47 20: 46 20: 47 21: 46 20 21: 47 22: 46 22: 47 23: 47 24: 47 25: 47 26: 47 27: 46 27: 47 28: 46 28: 47 29: 47 30: 46 30: 47 31: 46 31: 47 32: 46 32: 47 33: 46 33: 47 34: 46 34: 47 35: 46 35: 47 36: 46 36: 47 37: 21 37: 46 37: 47 37: 79 25 38: 21 39: 21 39: 79 40: 79 41: 21 41: 79 42: 21 42: 79 43: 79 44: 21 44: 79 45: 21 45: 79 46: 21 46: 79 47: 21 51: 43 There are 23 hits at base# 47 46 & 47 often together There are 17 hits at base# 46 There are 11 hits at base# 43 30 Sau96I Ggncc 70 1: 44 2: 3 2: 44 3: 44 4: 44 5: 3 5: 44 6: 44 7: 44 8: 22 8: 44 9: 44 10: 44 11: 3 12: 22 13: 22 14: 22 15: 33 15: 47 16: 47 17: 47 18: 47 19: 47 20: 47 21: 47 22: 47 23: 33 23: 47 24: 33 24: 47 25: 33 25: 47 35 26: 33 26: 47 27: 47 28: 47 29: 47 30: 47 31: 33 31: 47 32: 33 32: 47 33: 33 33: 47 34: 33 34: 47 35: 47 36: 47 37: 21 37: 22 37: 47 38: 21 38: 22 39: 21 39: 22 41: 21 41: 22 42: 21 42: 22 43: 80 44: 21 44: 22 45: 21 45: 22 46: 21 46: 22 47: 21 47: 22 50: 22 51: 44 - 111 There are 23 hits at base# 47 These do not occur together. There are 11 hits at base# 44 There are 14 hits at base# 22 These do occur together. There are 9 hits at base# 21 5 BsmAI GTCTCNnnnn 22 1: 58 3: 58 4: 58 5: 58 8: 58 9: 58 10: 58 13: 70 36: 18 37: 70 38: 70 39: 70 40: 70 41: 70 42: 70 44: 70 45: 70 46: 70 10 47: 70 48: 48 49: 48 50: 85 There are 11 hits at base# 70 -"- Nnnnnngagac 27 13: 40 15: 48 16: 48 17: 48 18: 48 20: 48 15 21: 48 22: 48 23: 48 24: 48 25: 48 26: 48 27: 48 28: 48 29: 48 30: 10 30: 48 31: 48 32: 48 33: 48 35: 48 36: 48 43: 40 44: 40 45: 40 46: 40 47: 40 There are 20 hits at base# 48 20 AvaII Ggwcc 44 Sau96I($M.HaeIII) Ggwcc 44 2: 3 5: 3 6: 44 8: 44 9: 44 10: 44 11: 3 12: 22 13: 22 14: 22 15: 33 15: 47 25 16: 47 17: 47 18: 47 19: 47 20: 47 21: 47 22: 47 23: 33 23: 47 24: 33 24: 47 25: 33 25: 47 26: 33 26: 47 27: 47 28: 47 29: 47 30: 47 31: 33 31: 47 32: 33 32: 47 33: 33 33: 47 34: 33 34: 47 35: 47 36: 47 37: 47 30 43: 80 50: 22 There are 23 hits at base# 47 44 & 47 never together There are 4 hits at base# 44 PpuMI RGgwccy 27 35 6: 43 8: 43 9: 43 10: 43 15: 46 16: 46 17: 46 18: 46 19: 46 20: 46 21: 46 22: 46 23: 46 24: 46 25: 46 26: 46 27: 46 28: 46 - 112 30: 46 31: 46 32: 46 33: 46 34: 46 35: 46 36: 46 37: 46 43: 79 There are 22 hits at base# 46 43 and 46 never occur together. There are 4 hits at base# 43 5 BsmFI GGGAC 3 8: 43 37: 46 50: 77 -I"- gtccc 33 15: 48 16: 48 17: 48 1: 0 1: 0 20: 48 10 21: 48 22: 48 23: 48 24: 48 25: 48 26: 48 27: 48 28: 48 29: 48 30: 48 31: 48 32: 48 33: 48 34: 48 35: 48 36: 48 37: 54 38: 54 39: 54 40: 54 41: 54 42: 54 43: 54 44: 54 45: 54 46: 54 47: 54 15 There are 20 hits at base# 48 There are 11 hits at base# 54 HinfI Gantc 80 8: 77 12: 16 13: 16 14: 16 15: 16 15: 56 20 15: 77 16: 16 16: 56 16: 77 17: 16 17: 56 17: 77 18: 16 18: 56 18: 77 19: 16 19: 56 19: 77 20: 16 20: 56 20: 77 21: 16 21: 56 21: 77 22: 16 22: 56 22: 77 23: 16 23: 56 23: 77 24: 16 24: 56 24: 77 25: 16 25: 56 25 25: 77 26: 16 26: 56 26: 77 27: 16 27: 26 27: 56 27: 77 28: 16 28: 56 28: 77 29: 16 29: 56 29: 77 30: 56 31: 16 31: 56 31: 77 32: 16 32: 56 32: 77 33: 16 33: 56 33: 77 34: 16 35: 16 35: 56 35: 77 36: 16 36: 26 30 36: 56 36: 77 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46 There are 34 hits at base# 16 35 TfiI Gawtc 21 8: 77 15: 77 16: 77 17: 77 18: 77 19: 77 20: 77 21: 77 22: 77 23: 77 24: 77 25: 77 26: 77 27: 77 28: 77 29: 77 31: 77 32: 77 - 113 33: 77 35: 77 36: 77 There are 21 hits at base# 77 MlyI GAGTC 38 5 12: 16 13: 16 14: 16 15: 16 16: 16 17: 16 18: 16 19: 16 20: 16 21: 16 22: 16 23: 16 24: 16 25: 16 26: 16 27: 16 27: 26 28: 16 29: 16 31: 16 32: 16 33: 16 34: 16 35: 16 36: 16 36: 26 37: 16 38: 16 39: 16 40: 16 10 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46 There are 34 hits at base# 16 -"- GACTC 21 15 15: 56 16: 56 17: 56 18: 56 19: 56 20: 56 21: 56 22: 56 23: 56 24: 56 25: 56 26: 56 27: 56 28: 56 29: 56 30: 56 31: 56 32: 56 33: 56 35: 56 36: 56 There are 21 hits at base# 56 20 Plel gagtc 38 12: 16 13: 16 14: 16 15: 16 16: 16 17: 16 18: 16 19: 16 20: 16 21: 16 22: 16 23: 16 24: 16 25: 16 26: 16 27: 16 27: 26 28: 16 25 29: 16 31: 16 32: 16 33: 16 34: 16 35: 16 36: 16 36: 26 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46 There are 34 hits at base# 16 30 -"- gactc 21 15: 56 16: 56 .17: 56 18: 56 19: 56 20: 56 21: 56 22: 56 23: 56 24: 56 25: 56 26: 56 27: 56 28: 56 29: 56 30: 56 31: 56 32: 56 33: 56 35: 56 36: 56 35 There are 21 hits at base# 56 AiwNI CAGNNNctg 26 15: 68 16: 68 12: 68 18: 68 19: 68 20: 68 - 114 21: 68 22: 68 23: 68 24: 68 25: 68 26: 68 27: 68 28: 68 29: 68 30: 68 31: 68 32: 68 33: 68 34: 68 35: 68 36: 68 39: 46 40: 46 41: 46 42: 46 5 There are 22 hits at base# 68 - 115 Table 8: Kappa FR1 GLGs !1 2 3 4 5 6 7 8 9 10 11 12 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT 13 14 15 16 17 18 19 20 21 22 23 5 GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 012 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC 02 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 018 10 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 08 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! A20 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT 15 GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC A30 AAC ATC CAG ATG ACC CAG TCT CCA TCT GCC ATG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT L14 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT Li 20 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! L15 GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L4 GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT 25 GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC L18 GAC ATC CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! L5 GAC ATC CAG ATG ACC CAG TCT CCA TCT TCT GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT L19 30 GAC ATC CAG TTG ACC CAG TCT CCA TCC TTC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC L8 GCC ATC CGG ATG ACC CAG TCT CCA TTC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L23 GCC ATC CGG ATG ACC CAG TCT CCA TCC TCA TTC TCT 35 GCA TCT ACA GGA GAC AGA GTC ACC ATC ACT TGT L9 - 116 GTC ATC TGG ATG ACC CAG TCT CCA TCC TTA CTC TCT GCA TCT ACA GGA GAC AGA GTC ACC ATC AGT TGT ! L24 GCC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! Lll 5 GAC ATC CAG ATG ACC CAG TCT CCT TCC ACC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L12 GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! 011 GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC 10 GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! 01 GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ! A17 GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ! Al 15 GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC ! A18 GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC ! A2 GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC 20 GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! A19 GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! A3 GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC TCA CCT GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ! A23 25 GAA ATT GTG TTG ACG CAG TCT CCA GGC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! A27 GAA ATT GTG TTG ACG CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! All GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT 30 GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC L2 GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L16 GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L6 35 GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT - 117 TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC L20 GAA ATT GTA ATG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L25 GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT 5 GTG TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC ! B3 GAA ACG ACA CTC ACG CAG TCT CCA GCA TTC ATG TCA GCG ACT CCA GGA GAC AAA GTC AAC ATC TCC TGC ! B2 GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC ! A26 10 GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC ! A10 GAT GTT GTG ATG ACA CAG TCT CCA GCT TTC CTC TCT GTG ACT CCA GGG GAG AAA GTC ACC ATC ACC TGC A14 118 Nl In , ' r- c C, 2 : ND N N %. N. N0 N N N Cq0 -4 ('4 C E0 rCr 0 0o 00!0 0 c I C, C, C, C' ' % C, ' % CS C 11 v 'r Cv Cv C In -0 r- co' *c' 0 N NN N F0 FO NF% A -1) Ins1 In, N4 ( N -n N- n 10 -- co e o A 5 0 0 CD CD ~~ 0 0 0 V4 N % IT~ Ln SiC '3 N co 4- 5-SC 5-C 5-5 -, a,' as 5- - '' m55 %,. No 00 0m 0* 0 0oN N s15 N In 3 N - C, F T' Ln C' 00 C' es C' '3In C% I" 3 fu 0 0 SC E- I I 5 LO) LO 119 0 0 C, 0 00 C, '0 .C '0 '0 L0 tr0 '0 ' , n C4 ~ ~ ~ (N (4 C4 N c4 C (N 11 C4N4 N 4 4 N C4eq I 0 C' 0 - ~ r 00( ~ %'. 0 10 -0 (N ID %0 10 C' 0 If, %0 1-co C '0 N C e4 C' C4C1 o~ !.. , e4 -n (N U, 0 ) (N-- (NN (N( N - 120 0o 00 0o T N' .4 C4 N C% C% C' C' C, E F LO'C '3- 121 C'4 * 0 xa f (N '3Ln W In 'n In'3 ' 'A 'n w~ '3 '3 ' 2 '3 '32 3 (C 1 J~~( C UC iC C U UC ~ C CC U C (4 In *rr '3%, r- 00 C, rn Un .1 I n n -n O 1 1 n I -) InAm r o C *~C C C ! ~C ~C C, C, In iC *' UI n IC ' r- c- C, Cu -t- - o -n co - >, -) 0 LO J 122 cN x ' ' ' ' 'D -In'4 cI N Ln 0 ('4 c1 - '3 Nq r4 c q N - C4J N C4 N N* r'4 Nl eq .1% ell l Inl 04' C N N 4 ('4 (' N c4' Ln AA In CI N 4 N (4 c4 Ln 1 (FZ() A -o (' (l - I' C4 C4 ('4 4 (Fl-- -A -* 123 x ' - 00 C, r- c C, In lol 1.CC% o C- Go C C, C vC' r- 00 C, N3 C4 A, C. C' f- 00 c' en In ~ (4N '3 U)* C1 1 wCC C4 ' C' C4C '3U' C':)0 3 3 ' - C'~ ~ '3 r-I ~ C 124 00

C

cc e C.4 V - Cc r0 Z C C,4 c- CDCCu > o o o -4 - 125 0 C0 oo C' C, 0 4 N ) . r n %0 I - N N NN cN N4 Nq N SA c N C% 0 0 0% C, A1 N4 N LO eq NT 'AZr o .',. *j* " C4 In -2 I- N 0 NO "- KK * 126 CD 0D 0 0 r4 A1 C14 C4 c4 In -n U' '- oD c' ,t ~.en em'3C U', fli en ~In U' 0n - N0' C' LO C

U'-

- 127 Table 10 Lambda FR1 GLG sequences VL1 CAG TCT GTG CTG ACT CAG CCA CCC TCG GTG TCT GAA GCC CCC AGG CAG AGG GTC ACC ATC TCC TGT ! la 5 cag tct gtg ctg acG cag ccG ccc tcA gtg tct gGG gcc ccA Ggg cag agg gtc acc atc tcc tgC ! le cag tct gtg ctg act cag cca ccc tcA gCg tct gGG Acc ccc Ggg cag agg gtc acc atc tcT tgt ! 1c cag tct gtg ctg act cag cca ccc tcA gCg tct gGG LO Acc ccc Ggg cag agg gtc acc atc tcT tgt ! g cag tct gtg Ttg acG cag ccG ccc tcA gtg tct gCG gcc ccA GgA cag aAg gtc acc atc tcc tgC lb VL2 CAG TCT GCC CTG ACT CAG CCT CCC TCC GCG TCC GGG L5 TCT CCT GGA CAG TCA GTC ACC ATC TCC TGC ! 2c cag tct gcc ctg act cag cct cGc tcA gTg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc! 2e cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc tcc tgc ! 2a2 20 cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc ! 2d cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc tcc tgc 2b2 VL3 ?5 TCC TAT GAG CTG ACT CAG CCA CCC TCA GTG TCC GTG TCC CCA GGA CAG ACA GCC AGC ATC ACC TGC! 3r tcc tat gag ctg act cag cca cTc tca gtg tcA gtg Gcc cTG gga cag acG gcc agG atT acc tgT 3j tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg 30 tcc cca gga caA acG gcc agG atc acc tgc! 3 p tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg tcc cTa gga cag aTG gcc agG atc acc tgc 3a tcT tCt gag ctg act cag GAC ccT GcT gtg tcT gtg Gcc TTG gga cag aca gTc agG atc acA tgc 31 - 128 'cc tat gTg ctg act cag cca ccc tca gtg tcA gtg Gcc cca gga Aag acG gcc agG atT acc tgT ! 3h tcc tat gag ctg acA cag cTa ccc tcG gtg tcA gtg tcc cca gga cag aca gcc agG atc acc tgc ! 3e tcc tat gag ctg aTG cag cca ccc tcG gtg tcA gtg tcc cca gga cag acG gcc agG atc acc tgc ! 3m tcc tat gag ctg acA cag cca Tcc tca gtg tcA gtg tcT ccG gga cag aca gcc agG atc acc tgc V2-19 VL4 CTG CCT GTG CTG ACT CAG CCC CCG TCT GCA TCT GCC TTG CTG GGA GCC TCG ATC AAG CTC ACC TGC ! 4c cAg cct gtg ctg act caA TcA TcC tct gcC tct gcT tCC ctg gga Tcc tcg Gtc aag ctc acc tgc ! 4a cAg cTt gtg ctg act caA TcG ccC tct gcC tct gcc 5 tCC ctg gga gcc tcg Gtc aag ctc acc tgc ! 4b VL5 CAG CCT GTG CTG ACT CAG CCA CCT TCC TCC TCC GCA TCT CCT GGA GAA TCC GCC AGA CTC ACC TGC ! 5e cag Gct gtg ctg act cag ccG Gct tcc CTc tcT gca 0 tct cct gga gCa tcA gcc agT ctc acc tgc ! 5c cag cct gtg ctg act cag cca Tct tcc CAT tcT gca tct Tct gga gCa tcA gTc aga ctc acc tgc ! 5b VL6 AAT TTT ATG CTG ACT CAG CCC CAC TCT GTG TCG GAG 5 TCT CCG GGG AAG ACG GTA ACC ATC TCC TGC ! 6a VL7 CAG ACT GTG GTG ACT CAG GAG CCC TCA CTG ACT GTG TCC CCA GGA GGG ACA GTC ACT CTC ACC TGT ! 7a cag Gct gtg gtg act cag gag ccc tca ctg act gtg 0 tcc cca gga ggg aca gtc act ctc acc tgt ! 7b VL8 CAG ACT GTG GTG ACC CAG GAG CCA TCG TTC TCA GTG TCC CCT GGA GGG ACA GTC ACA CTC ACT TGT ! 8a - 129 VL9 CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA TCA GCC TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC 9a VL10 5 CAG GCA GGG CTG ACT CAG CCA CCC TCG GTG TCC AAG GGC TTG AGA CAG ACC GCC ACA CTC ACC TGC ! 10a - 130 Table 11 RERSs found in human lambda FR1 GLGs ! There are 31 lambda GLGs MlyI NnnnnnGACTC 25 1: 6 3: 6 4: 6 6: 6 7: 6 8: 6 5 9: 6 10: 6 11: 6 12: 6 15: 6 16: 6 20: 6 21: 6 22: 6 23: 6 23: 50 24: 6 25: 6 25: 50 26: 6 27: 6 28: 6 30: 6 31: 6 There are 23 hits at base# 6 GAGTCNNNNNn 1 26: 34 MwoI GCNNNNNnngc 20 5 1: 9 2: 9 3: 9 4: 9 11: 9 11: 56 12: 9 13: 9 14: 9 16: 9 17: 9 18: 9 19: 9 20: 9 23: 9 24: 9 25: 9 26: 9 30: 9 31: 9 There are 19 hits at base# 9 0 HinfI Gantc 27 1: 12 3: 12 4: 12 6: 12 7: 12 8: 12 9: 12 10: 12 11: 12 12: 12 15: 12 16: 12 20: 12 21: 12 22: 12 23: 12 23: 46 23: 56 24: 12 25: 12 25: 56 26: 12 26: 34 27: 12 5 28: 12 30: 12 31: 12 There are 23 hits at base# 12 PleI gactc 25 1: 12 3: 12 4: 12 6: 12 7: 12 8: 12 9: 12 10: 12 11: 12 12: 12 15: 12 16: 12 0 20: 12 21: 12 22: 12 23: 12 23: 56 24: 12 25: 12 25: 56 26: 12 27: 12 28: 12 30: 12 31: 12 There are 23 hits at base# 12 5 -"- gagtc 1 - 131 26: 34 DdeI Ctnag 32 1: 14 2: 24 3: 14 3: 24 4: 14 4: 24 5 5: 24 6: 14 7: 14 7: 24 8: 14 9: 14 10: 14 11: 14 11: 24 12: 14 12: 24 15: 5 15: 14 16: 14 16: 24 19: 24 20: 14 23: 14 24: 14 25: 14 26: 14 27: 14 28: 14 29: 30 30: 14 31: 14 LO There are 21 hits at base# 14 BsaJI Ccnngg 38 1: 23 1: 40 2: 39 2: 40 3: 39 3: 40 4: 39 4: 40 5: 39 11: 39 12: 38 12: 39 L5 13: 23 13: 39 14: 23 14: 39 15: 38 16: 39 17: 23 17: 39 18: 23 18: 39 21: 38 21: 39 21: 47 22: 38 22: 39 22: 47 26: 40 27: 39 28: 39 29: 14 29: 39 30: 38 30: 39 30: 47 31: 23 31: 32 ?0 There are 17 hits at base# 39 There are 5 hits at base# 38 There are 5 hits at base# 40 Makes cleavage ragged. MnlI cctc 35 1: 23 2: 23 3: 23 4: 23 5: 23 6: 19 ?5 6: 23 7: 19 8: 23 9: 19 9: 23 10: 23 11: 23 13: 23 14: 23 16: 23 17: 23 18: 23 19: 23 20: 47 21: 23 21: 29 21: 47 22: 23 22: 29 22: 35 22: 47 23: 26 23: 29 24: 27 27: 23 28: 23 30: 35 30: 47 31: 23 30 There are 21 hits at base# 23 There are 3 hits at base# 19 There are 3 hits at base# 29 There are 1 hits at base# 26 There are 1 hits at base# 27 These could make cleavage ragged. 35 -"- gagg 7 - 132 1: 48 2: 48 3: 48 4: 48 27: 44 28: 44 29: 44 BssKI Nccngg 39 1: 40 2: 39 3: 39 3: 40 4: 39 4: 40 5: 39 6: 31 6: 39 7: 31 7: 39 8: 39 9: 31 9: 39 10: 39 11: 39 12: 38 12: 52 13: 39 13: 52 14: 52 16: 39 16: 52 17: 39 17: 52 18: 39 18: 52 19: 39 19: 52 21: 38 22: 38 23: 39 24: 39 26: 39 27: 39 28: 39 29: 14 29: 39 30: 38 There are 21 hits at base# 39 There are 4 hits at base# 38 There are 3 hits at base# 31 5 There are 3 hits at base# 40 Ragged BstNI CCwgg 30 1: 41 2: 40 5: 40 6: 40 7: 40 8: 40 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 J 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28: 40 29: 15 29: 40 30: 39 There are 17 hits at base# 40 There are 7 hits at base# 53 5 There are 4 hits at base# 39 There are 1 hits at base# 41 Ragged PspGI ccwgg 30 1: 41 2: 40 5: 40 6: 40 7: 40 8: 40 D 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28: 40 29: 15 29: 40 30: 39 There are 17 hits at base# 40 5 There are 7 hits at base# 53 - 133 There are 4 hits at base# 39 There are 1 hits at base# 41 ScrFI CCngg 39 5 1: 41 2: 40 3: 40 3: 41 4: 40 4: 41 5: 40 6: 32 6: 40 7: 32 7: 40 8: 40 9: 32 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 40 19: 53 21: 39 .0 22: 39 23: 40 24: 40 26: 40 27: 40 28: 40 29: 15 29: 40 30: 39 There are 21 hits at base# 40 There are 4 hits at base# 39 There are 3 hits at base# 41 5 MaeIII gtnac 16 1: 52 2: 52 3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 26: 52 27: 10 27: 52 28: 10 28: 52 29: 10 29: 52 30: 52 ?0 There are 13 hits at base# 52 Tsp45I gtsac 15 1: 52 2: 52 3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 27: 10 27: 52 28: 10 28: 52 ?5 29: 10 29: 52 30: 52 There are 12 hits at base# 52 HphI tcacc 26 1: 53 2: 53 3: 53 4: 53 5: 53 6: 53 30 7: 53 8: 53 9: 53 10: 53 11: 59 13: 59 14: 59 17: 59 18: 59 19: 59 20: 59 21: 59 22: 59 23: 59 24: 59 25: 59 27: 59 28: 59 30: 59 31: 59 There are 16 hits at base# 59 - 134 There are 10 hits at base# 53 BspMI ACCTGCNNNNn 14 11: 61 13: 61 14: 61 17: 61 18: 61 19: 61 5 20: 61 21: 61 22: 61 23: 61 24: 61 25: 61 30: 61 31: 61 There are 14 hits at base# 61 Goes into CDR1 - 135 Table 12: Matches to URE FR3 adapters in 79 human HC. A. List of Heavy-chains genes sampled AF008566 AF103367 HSA235674 HSU94417 S83240 AF035043 AF103368 HSA235673 HSU94418 SABVH169 5 AF103026 AF103369 HSA240559 HSU96389 SADEIGVH afl03033 AF103370 HSCB201 HSU96391 SAH2IGVH AF103061 afl03371 HSIGGVHC HSU96392 SDA3IGVH Afl03072 AF103372 HSU44791 HSU96395 SIGVHTTD afl03078 AF158381 HSU44793 HSZ93849 SUK4IGVH 10 AF103099 E05213 HSU82771 HSZ93850 AF103102 E05886 HSU82949 HSZ93851 AF103103 E05887 HSU82950 HSZ93853 AF103174 HSA235661 HSU82952 HS293855 AF103186 HSA235664 HSU82961 HSZ93857 15 afl03187 HSA235660 HSU86522 HSZ93860 AF103195 HSA235659 HSU86523 HSZ93863 afl03277 HSA235678 HSU92452 MCOMFRAA afl03286 HSA235677 HSU94412 MCOMFRVA AF103309 HSA235676 HSU94415 S82745 20 afl03343 HSA235675 HSU94416 S82764 Table 12B. Testing all distinct GLGs from bases 89.1 to 93.2 of the heavy variable domain Id Nb 0 1 2 3 4 SEQ ID NO: 25 1 38 15 11 10 0 2 Seql gtgtattactgtgc 25 2 19 7 6 4 2 0 Seq2 gtAtattactgtgc 26 3 1 0 0 1 0 0 Seq3 gtgtattactgtAA 27 4 7 1 5 1 0 0 Seq4 gtgtattactgtAc 28 5 0 0 0 0 0 0 Seq5 Ttgtattactgtgc 29 30 6 0 0 0 0 0 0 Seq6 TtgtatCactgtgc 30 7 3 1 0 1 1 0 Seq7 ACAtattactgtgc 31 8 2 0 2 0 0 0 Seq8 ACgtattactgtgc 32 9 9 2 2 4 1 0 Seq9 ATtattactqtqc 33 Group 26 26 21 4 2 35 Cumulative 26 52 73 77 79 Table 12C Most important URE recognition seqs in FR3 Heavy 1 VHSzyl GTGtattactgtgc (ONSHC103) (SEQ ID NO:25) 2 VHSzy2 GTAtattactgtgc (ONSHC323) (SEQ ID NO:26) 3 VHSzy4 GTGtattactgtac (ONSHC349) (SEQ ID NO:28) 40 4 VHSzy9 ATGtattactgtgc (ONSHC5a) (SEQ ID NO:33) Table 12D, testing 79 human HC V genes with four probes Number of sequences............ 79 Number of bases............... 29143 - 136 Number of mismatches Id Best 0 1 2 3 4 5 5 1 39 15 11 10 1 2 0 Seq1 gtgtattactgtgc (SEQ ID NO:25) 2 22 7 6 5 3 0 1 Seq2 gtAtattactgtgc (SEQ ID NO:26) 3 7 1 5 1 0 0 0 Seq4 gtgtattactgtAc (SEQ ID NO:28) 4 11 2 4 4 1 0 0 Seq9 ATqtattactqtqc (SEQ ID NO:33) Group 25 26 20 5 2 LO Cumulative 25 51 71 76 78 One sequence has five mismatches with sequences 2, 4, and 9; it is scored as best for 2. Id is the number of the adapter. Best is the number of sequence for which the identified 15 adapter was the best available. The rest of the table shows how well the sequences match the adapters. For example, there are 10 sequences that match VHSzyl(Id=l) with 2 mismatches and are worse for all other adapters. In this sample, 90% come within 2 bases of one of 20 the four adapters.

- 137 Table 13 The following list of enzymes was taken from http://rebase.neb.com/cgi-bin/asymmlist. I have removed the enzymes that a) cut within the recognition, b) cut on 5 both sides of the recognition, or c) have fewer than 2 bases between recognition and closest cut site. REBASE Enzymes 04/13/2001 10 Type II restriction enzymes with asymmetric recognition sequences: Enzymes Recognition Sequence Isoschizomers Suppliers AarI CACCTGCNNNN^NNNN y AceIII CAGCTCNNNNNNN^NNNN_ Bbr7I GAAGACNNNNNNN^NNNN 15 BbvI GCAGCNNNNNNNN^NNNN y BbvII GAAGACNN^NNNN Bce83I CTTGAGNNNNNNNNNNNNNN_NN^ BceAI ACGGCNNNNNNNNNNNN^NN

-

y BcefI ACGGCNNNNNNNNNNNN^N 20 BciVI GTATCCNNNNN N^ BfuI y BfiI ACTGGGNNNN N^ BmrI y BinI GGATCNNNN^N BscAI GCATCNNNN^NN BseRI GAGGAGNNNNNNNN NN^ y 25 BsmFI GGGACNNNNNNNNNN^NNNN_ BspLU11III y BspMI ACCTGCNNNN^NNNN_ Acc36I y EciI GGCGGANNNNNNNNN NN^ -'Y Eco57I CTGAAGNNNNNNNNNNNNNN_NN^ BspKT5I y FauI CCCGCNNNN^NN BstFZ438I y 30 FokI GGATGNNNNNNNNN^NNNN BstPZ418I y GsuI CTGGAGNNNNNNNNNNNNNN_NN^

-

y HgaI GACGCNNNNN^NNNNN y HphI GGTGANNNNNNN N^ AsuHPI y MboII GAAGANNNNNNN N^ y 35 MlyI GAGTCNNNNN^ SchI y MmeI TCCRACNNNNNNNNNNNNNNNNNN NN^ MnlI CCTCNNNNNN N^ y PleI GAGTCNNNN^N_ PpsI y RleAI CCCACANNNNNNNNN NNN^ 40 SfaNI GCATCNNNNN^NNNN_ BspST5I y SspD5I GGTGANNNNNNNN^ Sthl32I CCCGNNNN^NNNN StsI GGATGNNNNNNNNNN^NNNN_ TaqII GACCGANNNNNNNNNNN^, CACCCANNNNNNNNN_NN^ - 45 TthlllII CAARCANNNNNNNNNNN^ UbaPI CGAACG The notation is ^ means cut the upper strand and _ means cut the lower strand. If the upper and lower strand are cut at the same place, then only ^ appears.

138 u u roo o1 J 4 0) V ~ at.~ O41 I 'a 4-o (04- ma' mN - N 41 mi O4J (U 4 0) 001- 4-J 4J 0.'4-' - -4 a .- U J 'k E in i (D Aj 4j 0 ONO 0 u 0< u 0( E-0u .'-1 u 4-U 4J -) E-O1n 0) 0 0 u 0 E- 4) -) E- 4-)E- 4 UO 0) 00 ) u ~ON O C-- k U m m c ( m.~ 0 -) J m (a~'- (D (a' a (o ( (y '- E- E". a)' ON1 'aO - - m. Ur) U' (a (a' m m m m UON.-I a-' 01 mUN (O' 41 4J 4.) 4. 4J V U 0oa) 0ON4i ()U E- ~-E->O * 0 4J -% 41ON 4.)M 0 F-' (nO -' * - -'4 - u' C) ) 0 a u- ONU 0 )0 0 U ON E 4- ' M4J WO maCON (DD' &4 m J *a) -C) 1 'UC\U F. <F- < - m Ol - - -- UM4-MMi- I E- ~0t On El 0 - " E .--. 0-E- 00 0)<d OE'-0 .- 1 lU <U U 0 < U c E-' E- CM 4- ON m 0Xr ON (a 0 -'O U E- 41 r/) - 4J-40---H Or'E-~ .I I (D C:u4 a UL)U m l ~o:4 O - 1U>E-X M E- (1 - M U S E- XL LA ON 1 )0 - . t7 E-0 < Imco - =~ - I 4.~ ) (D <. 0 ODm CL > > > > In C0 LA - 139 Table 15: Use of FokI as "Universal Restriction Enzyme" FokI - for dsDNA, I represents sites of cleavage sites of cleavage 5'-cacGGATGtg--nnnnnnnlnnnnnnn-3'(SEQ ID NO:15) 5 3'-gtqCCTACac--nnnnnnnnnnninnn-5'(SEQ ID NO:16) RECOG NITion of FokI Case I 5'-...gtgltatt-actgtgc..Substrate....-3' (SEQ ID NO:17) 10 3'-cac-ataa l tqacacg--, _qtGTAGGcac\ 5'- caCATCCgtg/(SEQ ID NO:18) Case II 5'-...gtgtattlagac-tgc..Substrate....-3'(SEQ ID NO:19) 15 c-cacataa-tctg I acg-5' /gtgCCTACac \cacGGATGtg-3'(SEQ ID NO:20) Case III (Case I rotated 180 degrees) /gtgCCTACac-5' 20 \cacGGATGjg atatcttlacag-tcc-3' Adapter (SEQ ID NO:21) 3'-...cacagaa-tgtclagg..substrate....-5'(SEQ ID NO:22) Case IV (Case II rotated 180 degrees) 3'- gtGTAGGcac\ (SEQ ID NO:23) 25 r-c.aCATCCgtg/ 5'-gagitctc-actqaaoc Substrate 3'-...ctc-agagitgactcg...-5'(SEQ ID NO:24) Improved FokI adapters FokI - for dsDNA, I represents sites of cleavage 30 Case I Stem 11, loop 5, stem 11, recognition 17 5'-...catgtgltatt-actgtgc..Substrate....-3' 3'-gtacac-ataa L tgacac-, rT- _qtGTAGGcacG T 35 5'- caCATCCgtgc C

LTTJ

- 140 Case II Stem 10, loop 5, stem 10, recognition 18 5'-...gtgtattlagac-tgctgcc..Substrate....-3' rT, r-cacataa-tctgl acgacgg-5' 5 T gtgCCTACac C 'cacGGATGtg-3' LTTJ Case III (Case I rotated 180 degrees) Stem 11, loop 5, stem 11, recognition 20 10 r T, T TgtgCCTACac-5' G AcacGGATGt-, LTTJ qtqtcttlacag-tccattctg-3' Adapter 3'-...cacagaa-tgtclaggtaagac..substrate....-5' 15 Case IV (Case II rotated 180 degrees) Stem 11, loop 4, stem 11, recognition 17 rT, 3'- gtGTAGGcacc T r-.aCATCCgtgg T 20 5'-atcgagItctc-actqagc LTJ Substrate 3'-...tagctc-agagltgactcg...-5' BseRI I sites of cleavage 5'-cacGAGGAGnnnnnnnnnnInnnnn-3' 25 3'-gtgctcctcnnnnnnnn I nnnnnnn-5' RECOG NITion of BseRI Stem 11, loop 5, stem 11, recognition 19 3'-.......gaacatlcg-ttaagccagta.....5' 30 rT-T, cttgta-gclaattcggtcat-3' C GCTGAGGAGTC-J T cgactcctcag-5' An adapter for BseRI to cleave the substrate above.

LT---

141 *4 4J 01. ... -W 4J U 0 U U (T 0 T ON ON 0' m (0C m~~( (0 M 0 0 0' 0 " 44 4J 4~J 4~J 4! 4J 40' -W 0' 0'm 01t.0 U I. 4! 4-) 4 J 4! 4 moo~ ( Y a 'l0 $4 ) u uu - E-4E4U-4E-4 C14 En ON 0)J0) C t3 -4J~ ~ I 4 1-1O0U H E- - FO E- C m0 a, m0% m m m( 0 E-E-E-H -4 -4 - -4 $4 4 E - E- E- E- 4-i coI~ C m o-ocod a) 00.-oCC) CC) o 0 10 0G U UO E Uuo coE n(nHE fu = =*x0= = 0 0 - u r-C 0- CA -4 N C)0'' a) U H (Nc * 'I I I' Il I -'~ ~ : U (Nm eLO .4 44 4 4 . 0 a% .P 13 u 0 00 - ON -4 tyl a- .- 4 ) ~ a, (n -4 NN ty 0'l 0 --- - u a, co 41- . j4 j H 1 1U) ) a% (0 ( 0% % N cu =) '- r- ~ a r- 0 O C 4 MI I I --,4 c 'T 0D (NC 0'J-3-)44J 4-' Hu cu C. 60 v .IL C) 4- (O)LA ODJ' a% u g"eC9 9 0' (n wAf- cn 0LLLL m 4mr 1 4 1 14 1F -~ A 41mmm 4)% 'rMr M M 04 LAO C4 4J m = r- C) a 4~aa -4' 1 i m b OD 0 f) (7 D(n M) r- ) 05u u 0, z L )- N-z w- V' u" 142 Cu ~u -u u u VO u V v 00 u CC C CC% F. a

U--

143 -u u g-1 uL b Oo V~ ui LiE o c u u 0 - j w- ,- - Q~~. ci -i - 0 cl. C14~U ~ i 0 . L. 04 toi 2 0 2 ' u 8n W 0-u E tg 0 8i E vi -p 5 !! e up ( 0 0 Ol o. U 0 r4~~ ~ .- NJ(J 144 0 0 0 o 0 0 0 U) U U) I oo 0 0 0 6 C) C; c . 0 0 0 0 i-4 <I- - I < uf u 0 U 0 @0' V( u I Ch m F4 o~~ q. U.U t, E- I- ~ cc ' V~ 0 u IU to U 00 040 HuF4 'u ' 0 1-4 E- M A4 L o u ut .S) 0 r- - <- U E- U o U (j -, (N t- (_ o o ) u 4-) (V- ('- CN4 ("1)0 0 CAl 0 x C 0 1-45 0 UU g o u b :0 uo0 u u~ H q 0o om 0 Hu u 00 N~0 00t-CA 0 CIO On - uHt ! M ~ CL ~ v~ (D C T 0Z S -0 Co U o.Co ~ 0.. Go E z0 ,a c OtDC 1o01 0 1 u . q 0 C> c>00eq u I o. co 4) O or- r- Go E- E4 - 0 .0- - 1 C4 §u Mz*e LO C> O C) C - 146 What happens in the top strand: I site of cleavage in the upper strand (VL133-2a2*) 5'-g tctcc giga cag tcg aic 5 (VL133-31*) 5'-g gcc ttg g ga cag aca gic (VL133-2c*) 5'-g tct cCt g ga cag tCa gtc (VL1 33-1 c*) 5'-g gcc cca g I gg cag agg gtc 10 The following Extenders and Bridges all encode the AA sequence of 2a2 for codons 1-15 1 (ONLamExl33) 5'-ccTcTgAcTgAgT gcA cAg 15 !2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT 13 14 15 tcC ccG g! 2a2 20 1 1 (ONLamBI-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT 25 13 14 15 tcC ccG g ga cag teg at-3'! 2a2 N.B. the actual seq is the reverse complemcnt of the one shown. 30 (ONLamB2-133) [RC 5'-ccTcTgAcTgAgT gcA cAg 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT 35 13 14 15 tcC ccG g ga cag aca gt-3'! 31 N.B. the actual seq is the reverse complement of the one shown. 40 (ON LamB3-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg 2 3 4 5 6 7 8 9 10 11 12 45 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT 13 14 15 tcC ccG g ga cag tca gt -3'! 2c N.B. the actual seq is the reverse complement of the 50 ! one shown. !(ONLamB4-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg - - 147 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-s 5 13 14 15 tcC ccG g gg cag agg gt-3' ! 1c N.B. the actual seq is the reverse complement of the one shown. 10 (ONLam133PCR) 5'-ccTcTgAcTgAgT gcA cAg AGt gc-3' - 148 Table 19: Cleavage of 75 human light chains. Enzyme Recognition* Nch Ns Planned location of site AfeI AGCgct 0 0 AflII Cttaag 0 0 HC FR3 5 AgeI Accggt 0 0 AscI GGcgcgcc 0 0 After LC BglII Agatct 0 0 BsiWI Cgtacg 0 0 BspDI ATcgat 0 0 10 BssHII Gcgcgc 0 0 BstBI TTcgaa 0 0 DraIII CACNNNgtg 0 0 EagI Cggccg 0 0 FseI GGCCGGcc 0 0 15 FspI TGCgca 0 0 HpaI GTTaac 0 0 MfeI Caattg 0 0 HC FR1 MluI Acgcgt 0 0 NcoI Ccatgg 0 0 Heavy chain signal 20 NheI Gctagc 0 0 HC/anchor linker NotI GCggccgc 0 0 In linker after HC NruI TCGcga 0 0 PacI TTAATtaa 0 0 PmeI GTTTaaac 0 0 25 PmlI CACgtg 0 0 PvuI CGATcg 0 0 SacII CCGCgg 0 0 SalI Gtcgac 0 0 SfiI GGCCNNNNnggcc 0 0 Heavy Chain signal 30 SgfI GCGATcgc 0 0 SnaBI TACgta 0 0 StuI AGGcct 0 0 XbaI Tctaga 0 0 HC FR3 AatII GACGTc 1 1 35 AclI AAcgtt 1 1 AseI ATtaat 1 1 BsmI GAATGCN 1 1 BspEI Tccgga 1 1 HC FR1 BstXI CCANNNNNntgg 1 1 HC FR2 40 DrdI GACNNNNnngtc 1 1 HindIII Aagctt 1 1 PciI Acatgt 1 1 SapI gaagagc 1 1 ScaI AGTact 1 1 45 SexAI Accwggt 1 1 Spel Actagt 1 1 TliI Ctcgag 1 1 XhoI Ctcgag 1 1 BcgI cgannnnnntgc 2 2 50 BlpI GCtnagc 2 2 BssSI Ctcgtg 2 2 BstAPI GCANNNNntgc 2 2 EspI GCtnagc 2 2 KasI Ggcgcc 2 2 55 PflMI CCANNNNntgg 2 2 XmnI GAANNnnttc 2 2 ApaLI Gtgcac 3 3 LC signal seq - 149 NaeI GCCggc 3 3 NgoMI Gccggc 3 3 PvuII CAGctg 3 3 RsrII CGgwccg 3 3 5 BsrBI GAGcgg 4 4 BsrDI GCAATGNNn 4 4 BstZl7I GTAtac 4 4 EcoRI Gaattc 4 4 SphI GCATGc 4 4 10 SspI AATatt 4 4 AccI GTmkac 5 5 BclI Tgatca 5 5 BsmBI Nnnnnngagacg 5 5 BsrGI Tgtaca 5 5 15 DraI TTTaaa 6 6 NdeI CAtatg 6 6 HC FR4 SwaI ATTTaaat 6 6 BamHI Ggatcc 7 7 SacI GAGCTc 7 7 20 BciVI GTATCCNNNNNN 8 8 BsaBI GATNNnnatc 8 8 NsiI ATGCAt 8 8 Bsp120I Gggccc 9 9 CHl ApaI GGGCCc 9 9 CH1 25 PspOOMI Gggccc 9 9 BspHI Tcatga 9 11 EcoRV GATatc 9 9 AhdI GACNNNnngtc 11 11 BbsI GAAGAC 11 14 30 PsiI TTAtaa 12 12 BsaI GGTCTCNnnnn 13 15 XmaI Cccggg 13 14 AvaI Cycgrg 14 16 BglI GCCNNNNnggc 14 17 35 AlwNI CAGNNNctg 16 16 BspMI ACCTGC 17 19 XcmI CCANNNNNnnnntgg 17 26 BstEII Ggtnacc 19 22 HC FR4 Sse8387I CCTGCAgg 20 20 40 AvrII Cctagg' 22 22 HincII GTYrac 22 22 BsgI GTGCAG 27 29 MscI TGGcca 30 34 BseRI NNnnnnnnnnctcctc 32 35 45 Bsu36I CCtnagg 35 37 PstI CTGCAg 35 40 EciI nnnnnnnnntccgcc 38 40 PpuMI RGgwccy 41 50 StyI Ccwwgg 44 73 50 EcoOl09I RGgnccy 46 70 Acc65I Ggtacc 50 51 KpnI GGTACc 50 51 BpmI ctccag 53 82 AvaII Ggwcc 71 124 55 * cleavage occurs in the top strand after the last upper-case base. For REs that cut palindromic sequences, the lower strand is cut at the symmetrical site.

- 150 Table 20: Cleavage of 79 human heavy chains Enzyme Recognition Nch Ns Planned location of site Afel AGCgct 0 0 AflII Cttaag 0 0 HC FR3 5 AscI GGcgcgcc 0 0 After LC BsiWI Cgtacg 0 0 BspDI ATcgat 0 0 BssHII Gcgcgc 0 0 FseI GGCCGGcc 0 0 0 HpaI GTTaac 0 0 NheI Gctagc 0 0 HC Linker NotI GCggccgc 0 0 In linker, HC/anchor NruI TCGcga 0 0 NsiI ATGCAt 0 0 5 PacI TTAATtaa 0 0 PciI Acatgt 0 0 PmeI GTTTaaac 0 0 PvuI CGATcg 0 0 RsrII CGgwccg 0 0 10 SapI gaagagc 0 0 SfiI GGCCNNNNnggcc 0 0 HC signal seq SgfI GCGATcgc 0 0 SwaI ATTTaaat 0 0 AclI AAcgtt 1 1 '5 AgeI Accggt 1 1 AseI ATtaat 1 1 AvrII Cctagg 1 1 BsmI GAATGCN 1 1 BsrBI GAGcgg 1 1 0 BsrDI GCAATGNNn 1 1 DraI TTTaaa 1 1 FspI TGCgca 1 1 HindIII Aagctt 1 1 MfeI Caattg 1 1 HC FR1 15 NaeI GCCggc 1 1 NgoMI Gccggc 1 1 SpeI Actagt 1 1 Acc65I Ggtacc 2 2 BstBI TTcgaa 2 2 .0 KpnI GGTACc 2 2 MluI Acgcgt 2 2 NcoI Ccatgg 2 2 In HC signal seq NdeI CAtatg 2 2 HC FR4 PmlI CACgtg 2 2 5 XcmI CCANNNNNnnnntgg 2 2 BcgI cgannnnnntgc 3 3 BclI Tgatca 3 3 BglI GCCNNNNnggc 3 3 BsaBI GATNNnnatc 3 3 0 BsrGI Tgtaca 3 3 SnaBI TACgta 3 3 Sse8387I CCTGCAgg 3 3 ApaLI Gtgcac 4 4 LC Signal/FR1 BspHI Tcatga 4 4 5 BssSI Ctcgtg 4 4 PsiI TTAtaa 4 5 - 151 SphI GCATGc 4 4 AhdI GACNNNnngtc 5 5 BspEI Tccgga 5 5 HC FR1 MscI TGGcca 5 5 5 SacI GAGCTc 5 5 ScaI AGTact 5 5 SexAI Accwggt 5 6 SspI AATatt 5 5 TliI Ctcgag 5 5 10 Xhol Ctcgag 5 5 BbsI GAAGAC 7 8 BstAPI GCANNNNntgc 7 8 BstZl7I GTAtac 7 7 EcoRV GATatc 7 7 15 EcoRI Gaattc 8 8 BlpI GCtnagc 9 9 Bsu36I CCtnagg 9 9 DraIII CACNNNgtg 9 9 EspI GCtnagc 9 9 20 StuI AGGcct 9 13 XbaI Tctaga 9 9 HC FR3 Bspl20I Gggccc 10 11 CH1 ApaI GGGCCc 10 11 CH1 PspOOMI Gggccc 10 11 25 BciVI GTATCCNNNNNN 11 11 SalI Gtcgac 11 12 DrdI GACNNNNnngtc 12 12 KasI Ggcgcc 12 12 XmaI Cccggg 12 14 30 BglII Agatct 14 14 HincII GTYrac 16 18 BamHI Ggatcc 17 17 PflMI CCANNNNntgg 17 18 BsmBI Nnnnnngagacg 18 21 35 BstXI CCANNNNNntgg 18 19 HC FR2 XmnI GAANNnnttc 18 18 SacII CCGCgg 19 19 PstI CTGCAg 20 24 PvuII CAGctg 20 22 40 AvaI Cycgrg 21 24 EagI Cggccg 21 22 AatII GACGTc 22 22 BspMI ACCTGC 27 33 AccI GTmkac 30 43 45 StyI Ccwwgg 36 49 AlwNI CAGNNNctg 38 44 BsaI GGTCTCNnnnn 38 44 PpuMI RGgwccy 43 46 BsgI GTGCAG 44 54 50 BseRI NNnnnnnnnnctcctc 48 60 EciI nnnnnnnnntccgcc 52 57 BstEII Ggtnacc 54 61 HC Fr4, 47/79 have one EcoO109I RGgnccy 54 86 BpmI ctccag 60 121 55 AvaII Ggwcc 71 140 - 152 Table 21: MALIA3, annotated MALIA3 9532 bases 1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc 5 gene ii continued 49 cca aat gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta 97 tct aat ggt caa act aaa tct act cgt tcg cag aat tgg gaa tca act 145 gtt aca tgg aat gaa act tcc aga cac cgt act tta gtt gca tat tta 193 aaa cat gtt gag cta cag cac cag att cag caa tta agc tct aag cca LO 241 tcc gca aaa.atg acc tct tat caa aag gag caa tta aag gta ctc tct 289 aat cct gac ctg ttg gag ttt gct tcc ggt ctg gtt cgc ttt gaa gct 337 cga att aaa acg cga tat ttg aag tct ttc ggg ctt cct ctt aat ctt 385 ttt gat gca atc cgc ttt gct tct gac tat aat agt cag ggt aaa gac 433 ctg att ttt gat tta tgg tca ttc tcg ttt tct gaa ctg ttt aaa gca 15 481 ttt gag ggg gat tca ATG aat att tat gac gat tcc gca gta ttg gac RBS?...... Start gene x, ii continues 529 gct atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct 577 ttt gca aaa gcc tct cgc tat ttt ggt ttt tat cgt cgt ctg gta aac 625 gag ggt tat gat agt gtt gct ctt act atg cct cgt aat tcc ttt tgg 20 673 cgt tat gta tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg 721 atg aat ctt tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att 769 aac gta gat ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt 817 ctt aaa atc gca TAA ! End X & II 25 832 ggtaattca ca ! M1 ES Q10 T15 843 ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt ! Start gene V 30 ! ! S17 S20 P25 E30 891 tct ggt gtt tct cgt cag ggc aag cct tat tca ctg aat gag cag ctt V35 E40 V45 35 939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ctt gtc aag att act D50 A55 L60 987 ctt gat gaa ggt cag cca gcc tat gcg cct ggt cTG TAC Acc gtt cat BsrGI... 40 L65 V70 S75 R80 1035 ctg tcc tct ttc aaa gtt ggt cag ttc ggt tcc ctt atg att gac cgt P85 K87 end of V 1083 ctg cgc ctc gtt ccg gct aag TAA C 45 1108 ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg ! Start gene VII 1150 ata caa atc tcc gtt gta ctt tgt ttc gcg ctt ggt ata atc 50 VII and IX overlap. ..... S2 V3 L4 V5 S10 1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttc gcc tct ttc gtt End VII 55 Istart IX L13 Wis G20 T25 E29 1242 tta ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta atg gaa - 153 1293 act tcc tc .... stop of IX, IX and VIII overlap by four bases 5 1301 ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct acc ctc Start signal sequence of viii. 1349 gtt ccg atg ctg tct ttc gct gct gag ggt gac gat ccc gca aaa gcg mature VIII --- > 10 1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat gcg 1445 tgg gcg atg gtt gtt gtc att 1466 gtc ggc gca act atc ggt atc aag ctg ttt aag 1499 aaa ttc acc tcg aaa gca ! 1515 .. ........... -35 15 1517 agc tga taaaccgat acaattaaag gctccttttg ..... -10 ... 1552 gagccttttt ttttGGAGAt ttt ! S.D. underlined 20 ! <------ III signal sequence ----------------------------- > M K K L L F A I P L V 1575 caac GTG aaa aaa tta tta ttc gca att cct tta gtt ! 1611 25 V P F Y S H S A Q 1612 gtt cct ttc tat tct cac aGT gcA Cag tCT ApaLI... 1642 GTC GTG ACG CAG CCG CCC TCA GTG TCT GGG GCC CCA GGG CAG 30 AGG GTC ACC ATC TCC TGC ACT GGG AGC AGC TCC AAC ATC GGG GCA BstEII... 1729 GGT TAT GAT GTA CAC TGG TAC CAG CAG CTT CCA GGA ACA GCC CCC AAA 1777 CTC CTC ATC TAT GGT AAC AGC AAT CGG CCC TCA GGG GTC CCT GAC CGA 1825 TTC TCT GGC TCC AAG TCT GGC ACC TCA GCC TCC CTG GCC ATC ACT 35 1870 GGG CTC CAG GCT GAG GAT GAG GCT GAT TAT 1900 TAC TGC CAG TCC TAT GAC AGC AGC CTG AGT 1930 GGC CTT TAT GTC TTC GGA ACT GGG ACC AAG GTC ACC GTC BstEII... 1969 CTA GGT CAG CCC AAG GCC AAC CCC ACT GTC ACT 40 2002 CTG TTC CCG CCC TCC TCT GAG GAG CTC CAA GCC AAC AAG GCC ACA CTA 2050 GTG TGT CTG ATC AGT GAC TTC TAC CCG GGA GCT GTG ACA GTG GCC TGG 2098 AAG GCA GAT AGC AGC CCC GTC AAG GCG GGA GTG GAG ACC ACC ACA CCC 2146 TCC AAA CAA AGC AAC AAC AAG TAC GCG GCC AGC AGC TAT CTG AGC CTG 2194 ACG CCT GAG CAG TGG AAG TCC CAC AGA AGC TAC AGC TGC CAG GTC ACG 45 2242 CAT GAA GGG AGC ACC GTG GAG AAG ACA GTG GCC CCT ACA GAA TGT TCA 2290 TAA TAA ACCG CCTCCACCGG GCGCGCCAAT TCTATTTCAA GGAGACAGTC ATA AscI..... PelB signal----------------------------------------------> 50 M K Y L L P T A A A G L L L L 2343 ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC 16 17 18 19 20 21 22 A A Q P A M A 55 2388 gcG GCC cag ccG GCC atq qcc SfiI............. NgoMI... (1/2) NcoI.........

- 154 FR1(DP47/V3-23)--------------- 23 24 25 26 27 28 29 30 E V Q L L E S G 5 2409 gaalgtt!CAAITTGittalgagltctlggt| I MfeI I --------------FR1------------------------------------------- 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 .0 G G L V Q P G G S L R L S C A 2433 IggclggtcttlgttlcaglcctlggtlggtltctlttalcgtlcttltctItgclgctI ---- FR1----------------->1 ... CDR1................ I --- FR2----- 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 .5 A S G F T F S S Y A M S W V R 2478 gctiTCCIGGAIttclactIttcltctItCGITAClGctlatgltctitgglgtticgC I BspEI I I BsiWII IBstXI. ------- FR2----------------------------------> ... CDR2......... 0 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Q A P G K G L E W V S A I S G 2523 ICAalgctlccTIGGtlaaalggtlttglgagltgglgttltctIgctlatcltctiggt| ... BstXI !5 ..... CDR2............................................I---FR3-- 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 S G G S T Y Y A D S V K G R F 2568 itctlggtiggclagtlactitacitat!gctIgacltcclgttlaaaIggtlegclttcI 30 -------- FR3------------------------------------------------- 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 T I S R D N S K N T L Y L Q M 2613 |actlatc|TCTIAGAlgaclaacItctIaaglaatiactictcItacIttgIcaglatgI 35 | XbaI I ---FR3----------------------------------------------------->1 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 N S L R A E D T A V Y Y C A K 10 2658 laaclagCITTAIAGgIgctIgagIgacIaCTIGCAIGtcItacitat Itgclgctlaaal AflI I I PstI I ....... CDR3.................I----FR4------------------------ 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 15 D Y E G T G Y A F D I W G Q G 2703 IgacltatigaalggtlactlggtitatlgctlttclgaCIATAITGglggtlcaalggtI I NdeI I (1/4) ---------------- FR4---------->1 50 136 137 138 139 140 141 142 T M V T V S S 2748 lactlatGIGTCIACCIgtcltctlagt I BstEII 1 From BstEII onwards, pV323 is same as pCES1, except as noted. 55 !BstEII sites may occur in light chains; not likely to be unique in final vector.

- 155 143 144 145 146 147 148 149 150 151 152 A S T K G P S V F P 2769 gcc tcc acc aaG GGC CCa tcg GTC TTC ccc 5 Bspl20I. BbsI... (2/2) ApaI.... 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 L A P S S K S T S G G T A A L 10 2799 ctg gca ccC TCC TCc aag agc acc tot ggg ggc aca gcg gcc ctg BseRI... (2/2) 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 G C L V K D Y F P E P V T V S 15 2844 ggc tgc ctg GTC AAG GAC TAC TTC CCc gaA CCG GTg acg gtg tcg AgeI.... 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 W N S G A L T S G V H T F P A 20 2889 tgg aac tca GGC GCC ctg acc agc ggc gtc cac acc ttc ccg gct KasI...(1/4) 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 V L Q S S G L Y S L S S V V T 25 2934 gtc cta cag tCt agc GGa ctc tac tcc ctc agc agc gta gtg acc (Bsu36I...) (knocked out) 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 V P S S S L G T Q T Y I C N V 30 2979 gtg ccC tCt tct agc tTG Ggc acc cag acc tac atc tgc aac gtg (BstXI...........)N.B. destruction of BstXI & BpmI sites. 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 N H K P S N T K V D K K V E P 35 3024 aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 243 244 245 K S C A A A H H H H H H S A 3069 aaa tct tgt GCG GCC GCt cat cac cac cat cat cac tct gct 40 NotI...... E Q K L I S E E D L N G A A 3111 gaa caa aaa ctc atc tca gaa gag gat ctg aat ggt gcc gca 45 D I N D D R M A S G A 3153 GAT ATC aac gat gat cgt atg gct AGC ggc gcc rEK cleavage site............ NheI... KasI... EcoRV.. 50 Domain 1 ----------------------------------------------------------- A E T V E S C L A 3183 gct gaa act gtt gaa agt tgt tta gca 55 ! K P H T E I S F 3210 aaa ccc cat aca gaa aat tca ttt ! T N V W K D D K T 60 3234 aCT AAC GTC TGG AAA GAC GAC AAA Act - 156 L D R Y A N y E G C L W N A T G V 3261 tta gat cgt tac gct aac tat gag ggt tgt ctg tgG AAT GCt aca ggc gtt BsmI 5 V V C T G D E T Q C Y G T W V P I 3312 gta gtt tgt act ggt GAC GAA ACT CAG TGT TAC GGT ACA TGG GTT cct att G L A I P E N LO 3363 ggg ctt gct atc cct gaa aat Li linker ----------------------------------- E G G G S E G G G S 3384 gag ggt ggt ggc tct gag ggt ggc ggt tct 15 E G G G S E G G G T 3414 gag ggt ggc ggt tct gag ggt ggc ggt act Domain 2 ----------------------------------- 20 3444 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat atc aac 3495 cct ctc gac ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct 3546 aat cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag aat BseRI 3597 aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc act 25 3645 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct 3693 gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt aaa ttC AGA AlwNI 3741 GAC TGc gct ttc cat tct ggc ttt aat gaa gat cca ttc gtt tgt gaa AlwNI 30 3789 tat caa ggc caa tcg tct gac ctg cct caa cct cct gtc aat gct 3834 ggc ggc ggc tct start L2 ------------------------------------------------------------ 3846 ggt ggt ggt tct 35 3858 ggt ggc ggc tct 3870 gag ggt ggt ggc tct gag ggt ggc ggt tct 3900 gag ggt ggc ggc tct gag gga ggc ggt tcc 3930 ggt ggt ggc tct ggt ! end L2 40 Domain 3 ------------------------------------------------------------- S G D F D Y E K M A N A N K G A 3945 tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct M T E N A D E N A L Q S D A K G 45 3993 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc K L D S V A T D Y G A A I D G F 4041 aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc 50 ! I G D V S G L A N G N G A T G D 4089 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat ! F A G S N S Q M A Q V G D G D N 55 !4137 ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat ! S P L M N N F R Q Y L P S L P Q 4185 tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa - 157 S V E C R P F V F S A G K P Y E 4233 tcg gtt gaa tgt cgc cct ttt gtc ttt agc gct ggt aaa cca tat gaa 5 F S I D C D K I N L F R 4281 ttt tct att gat tgt gac aaa ata aac tta ttc cgt End Domain 3 G V F A F L L Y V A T F M Y V F140 10 4317 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt start transmembrane segment S T F A N I L 4365 tct acg ttt gct aac ata ctg 15 R N K E S 4386 cgt aat aag gag tct TAA ! stop of iii Intracellular anchor. 20 Ml P2 V L L5 G I P L L10 L R F L G15 4404 tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc ctc ggt Start VI 4451 ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag 25 4499 ggc ttc ggt aag ata gct att gct att tca ttg ttt ctt get ctt att 4547 att ggg ctt aac tca att ctt gtg ggt tat ctc tct gat att agc gct 4595 caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att ctc ccg tct 4643 aat gcg ctt ccc tgt ttt tat gtt att ctc tct gta aag gct gct att 4691 ttc att ttt gac gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat 30 Ml A2 V3 F5 L1O G13 4739 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga end VI Start gene I 35 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 K T L V S V G K I Q D K I V A 4785 aag acg ctc gtt agc gtt ggt aag att cag gat aaa att gta gct ! 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 40 G C K I A T N L D L R L Q N L 4830 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac ctc ! 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 ! P Q V G R F A K T P R V L R I 45 4875 ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 P D K P S I S 0 L L A I G R G 4920 ccg gat aag cct tct ata tct gat ttg ctt get att ggg cgc ggt 50 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 ! N D S Y D E N K N G L L V L D 4965 aat gat tcc tac gat gaa aat aaa aac ggc ttg ctt gtt etc gat 55 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 ! E C G T W F N T R S W N D K E 5010 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa - 158 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 R Q P I I D W F L H A R K L G 5055 aga cag ccg att att gat tgg ttt cta cat gct cgt aaa tta gga 5 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 W D I I F L V Q D L S I V D K 5100 tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 10 Q A R S A L A E H V V Y C R R 5145 cag gcg cgt tct gca tta gct gaa cat gtt gtt tat tgt cgt cgt 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 L D R I T L P F V G T L Y S L 15 5190 ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 I T G S K M P L P K L H V G V 5235 att act ggc tcg aaa atg cct ctg cct aaa tta cat gtt ggc gtt 20 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 V K Y G D S Q L S P T V E R W 5280 gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg 25 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 L Y T G K N L Y N A Y D T K Q 5325 ctt tat act ggt aag aat ttg tat aac gca tat gat act aaa cag 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 30 A F S S N Y D S G V Y S Y L T 5370 gct ttt tct agt aat tat gat tcc ggt gtt tat tct tat tta acg 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 P Y L S H G R Y F K P L N L G 35 5415 cct tat tta tca cac ggt cgg tat ttc aaa cca tta aat tta ggt 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 ! Q K M K L T K I Y L K K F S R 5460 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc 40 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 V L C L A I G F A S A F T Y S 5505 gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt aca tat agt 45 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 Y I T Q P K P E V K K V V S Q 5550 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 50 T Y D F D K F T I D S S Q R L 5595 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 N L S Y R Y V F K D S K G K L 55 5640 aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA PacI - 159 ! 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 ! I N S D D L Q K Q G Y S L T Y 5685 ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat ! PacI 5! ! 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 ! iI D L C T V S I K K G N S N E ! iv Ml K 5730 att gat tta tgt act gtt tcc att aaa aaa ggt aat tca aAT Gaa 10 Start IV 344 345 346 347 348 349 i I V K C N .End of I iv L3 L N5 V 17 N F V10 15 5775 att gtt aaa tgt aat TAA T TTT GTT IV continued..... 5800 ttc ttg atg ttt gtt tca tca tct tct ttt gct cag gta att gaa atg 5848 aat aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca 5896 ggc gaa tcc gtt att gtt tct ccc gat gta aaa ggt act gtt act gta 20 5944 tat tca tct gac gtt aaa cct gaa aat cta cgc aat ttc ttt att tct 5992 gtt tta cgt gct aat aat ttt gat atg gtt ggt tca att cct tcc ata 6040 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca 6088 tca tct gat aat cag gaa tat gat gat aat tcc gct cct tct ggt ggt 6136 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat 25 6184 aac gtt egg gca aag gat tta ata cga gtt gtc gaa ttg ttt gta aag 6232 tct aat act tct aaa tcc tca aat gta tta tct att gac ggc tct aat 6280 cta tta gtt gtt TCT gca cct aaa gat att tta gat aac ctt cct caa ApaLI removed 6328 ttc ctt tct act gtt gat ttg cca act gac cag ata ttg att gag gqt 30 6376 ttg ata ttt gag gtt cag caa ggt gat got tta gat ttt tca ttt got 6424 gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc 6472 ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat 6520 ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat 6568 tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag aag 35 6616 ggt tct atc tct gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg MscI 6664 act ggt gaa tct gcc aat gta aat aat cca ttt cag acg att gag cgt 6712 caa aat gta ggt att tcc atg agc gtt ttt cct gtt gca atg gct ggc 6760 ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct 40 6808 tct act cag gca agt gat gtt att act aat caa aga agt att got aca 6856 acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggc ctc act 6904 gat tat aaa aac act tct caa gat tct ggc gta ccg ttc ctg tct aaa 6952 atc cct tta atc ggc ctc ctg ttt agc tcc cgc tct gat tcc aac gag 7000 gaa agc acg tta tac gtg ctc gtc aaa gca acc ata gta cgc gcc ctg 45 7048 TAG cggcgcatt End IV 7060 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 7120 gcccgctcct ttcgctttet tcccttcctt tctcgccacg ttcGCCGGCt ttccccgtca NgoMI 50 7180 agetetaaat egggggctcc ctttagggtt ccgatttagt getttacgge acctogacco 7240 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt DraIII 7300 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac DrdI 55 7360 aacacteaac cctatetogg getattettt tgatttataa gggattttge cgatttcgga 7420 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 7480 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga PvuII. BsmBI. 7540 aaaaccaccc tGGATCC AAGCTT 60 BamHI HindIII (4) - 160 Insert carrying bla gene 7563 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc 7600 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct BciVI 5 7660 gataaatgct tcaataatat tgaaaaAGGA AGAgt RBS.?... Start bla gene 7695 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt 7746 tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat gct .0 7797 gaa gat cag ttg ggC gCA CGA Gtg ggt tac atc gaa ctg gat ctc aac agc BssSI... ApaLI removed 7848 ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc 7899 act ttt aaa gtt ctg cta tgt cat aca cta tta tcc cgt att gac gcc ggg .5 7950 caa gaG CAA CTC GGT CGc cgg gcg cgg tat tct cag aat gac ttg gtt gAG BcgI ScaI 8001 TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa ScaI 8052 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta ctt 8103 ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg Pvul 8154 ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc 8205 ata cca aac gac gag cgt gac acc acg atg cct gta gca atg cca aca acg 8256 tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct tcc cgg caa caa 5 !FspI.... 8307 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt c-g cgc tng 8358 GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt ! BglI 30 8409 gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt ! BsaI 8460 atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat AhdI 8511 aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg TAA ctgt 35 stop 8560 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 8620 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 8680 cgttccactg tacgtaagac cccc 8704 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct 10 HindIII SalI.. (2/2) HincII 8740 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt 8790 CCTGAGG 15 Bsu36I 8797 ccgat actgtcgtcg tcccctcaaa ctggcagatg 8832 cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg 8892 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc 8952 tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg 50 9012 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaATTTAAA SwaI... 9072 Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat RBS? 9131 ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc 55 Start gene II 9182 tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata BglII... 9233 gct acc ctc tcc ggc atg aat tta tca gct aga acg gtt gaa tat cat att - 161 9284 gat ggt gat ttg act gtc tcc ggc ctt tct cac cct ttt gaa tct tta cct 9335 aca cat tac tca ggc att gca ttt aaa ata tat gag ggt tct aaa aat ttt 9386 tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt cat 9437 aat gtt ttt ggt aca acc gat tta gct tta tgc tct gag gct tta ttg ctt 5 9488 aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt ! 9532 gene II continues - 162 Table 21B: Sequence of MALIA3, condensed LOCUS MALIA3 9532 CIRCULAR ORIGIN 1 AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT 5 61 ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT 121 CGTTCGCAGA ATTGGGAATC AACTGTTACA TGGAATGAAA CTTCCAGACA CCGTACTTTA 181 GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA 241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG 361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT LO 421 CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA 481 TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT 541 AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT 601 GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT 661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG L5 721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT 781 TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA 841 CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT 901 CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG 961 AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC 0 1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC 1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT 1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT 1201 CAAAGATGAG TGTTTTAGTG TATTCTTTCG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA 1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT 5 1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA 1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA 1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA 1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT 1561 TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC 30 1621 TATTCTCACA GTGCACAGTC TGTCGTGACG CAGCCGCCCT CAGTGTCTGG GGCCCCAGGG 1681 CAGAGGGTCA CCATCTCCTG CACTGGGAGC AGCTCCAACA TCGGGGCAGG TTATGATGTA 1741 CACTGGTACC AGCAGCTTCC AGGAACAGCC CCCAAACTCC TCATCTATGG TAACAGCAAT 1801 CGGCCCTCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT CTGGCACCTC AGCCTCCCTG 1861 GCCATCACTG GGCTCCAGGC TGAGGATGAG GCTGATTATT ACTGCCAGTC CTATGACAGC 35 1921 AGCCTGAGTG GCCTTTATGT CTTCGGAACT GGGACCAAGG TCACCGTCCT AGGTCAGCCC 1981 AAGGCCAACC CCACTGTCAC TCTGTTCCCG CCCTCCTCTG AGGAGCTCCA AGCCAACAAG 2041 GCCACACTAG TGTGTCTGAT CAGTGACTTC TACCCGGGAG CTGTGACAGT GGCCTGGAAG 2101 GCAGATAGCA GCCCCGTCAA GGCGGGAGTG GAGACCACCA CACCCTCCAA ACAAAGCAAC 2161 AACAAGTACG CGGCCAGCAG CTATCTGAGC CTGACGCCTG AGCAGTGGAA GTCCCACAGA 40 2221 AGCTACAGCT GCCAGGTCAC GCATGAAGGG AGCACCGTGG AGAAGACAGT GGCCCCTACA 2281 GAATGTTCAT AATAAACCGC CTCCACCGGG CGCGCCAATT CTATTTCAAG GAGACAGTCA 2341 TAATGAAATA CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCG GCCCAGCCGG 2401 CCATGGCCGA AGTTCAATTG TTAGAGTCTG GTGGCGGTCT TGTTCAGCCT GGTGGTTCTT 2461 TACGTCTTTC TTGCGCTGCT TCCGGATTCA CTTTCTCTTC GTACGCTATG TCTTGGGTTC 45 2521 GCCAAGCTCC TGGTAAAGGT TTGGAGTGGG TTTCTGCTAT CTCTGGTTCT GGTGGCAGTA 2581 CTTACTATGC TGACTCCGTT AAAGGTCGCT TCACTATCTC TAGAGACAAC TCTAAGAATA 2641 CTCTCTACTT GCAGATGAAC AGCTTAAGGG CTGAGGACAC TGCAGTCTAC TATTGCGCTA 2701 AAGACTATGA AGGTACTGGT TATGCTTTCG ACATATGGGG TCAAGGTACT ATGGTCACCG 2761 TCTCTAGTGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC TCCAAGAGCA 50 2821 CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC GAACCGGTGA 2881 CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTCCA CACCTTCCCG GCTGTCCTAC 2941 AGTCTAGCGG ACTCTACTCC CTCAGCAGCG TAGTGACCGT GCCCTCTTCT AGCTTGGGCA 3001 CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG GACAAGAAAG 3061 TTGAGCCCAA ATCTTGTGCG GCCGCTCATC ACCACCATCA TCACTCTGCT GAACAAAAAC 55 3121 TCATCTCAGA AGAGGATCTG AATGGTGCCG CAGATATCAA CGATGATCGT ATGGCTGGCG 3181 CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG 3241 TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGTTGT CTGTGGAATG - 163 3301 CTACAGGCGT TGTAGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA TGGGTTCCTA 3361 TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGGTGGCGGT TCTGAGGGTG 3421 GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT 3481 ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA 5 3541 ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA 3601 GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG CACTGTTACT CAAGGCACTG 3661 ACCCCGTTAA AACTTATTAC CAGTACACTC CTGTATCATC AAAAGCCATG TATGACGCTT 3721 ACTGGAACGG TAAATTCAGA GACTGCGCTT TCCATTCTGG CTTTAATGAA GATCCATTCG 3781 TTTGTGAATA TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG 10 3841 GCTCTGGTGG TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG CTCTGAGGGT GGCGGTTCTG 3901 AGGGTGGCGG CTCTGAGGGA GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT GATTTTGATT 3961 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC 4021 TACAGTCTGA CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG 4081 ATGGTTTCAT TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG 15 4141 CTGGCTCTAA TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA 4201 ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTA 4261 GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG 4321 TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA 4381 TACTGCGTAA TAAGGAGTCT TAATCATGCC AGTTCTTTTG GGTATTCCGT TATTATTGCG 20 4441 TTTCCTCGGT TTCCTTCTGG TAACTTTGTT CGGCTATCTG CTTACTTTTC TTAAAAAGGG 4501 CTTCGGTAAG ATAGCTATTG CTATTTCATT GTTTCTTGCT CTTATTATTG GGCTTAACTC 4561 AATTCTTGTG GGTTATCTCT CTGATATTAG CGCTCAATTA CCCTCTGACT TTGTTCAGGG 4621 TGTTCAGTTA ATTCTCCCGT CTAATGCGCT TCCCTGTTTT TATGTTATTC TCTCTGTAAA 4681 GGCTGCTATT TTCATTTTTG ACGTTAAACA AAAAATCGTT TCTTATTTGG ATTGGGATAA 25 4741 ATAATATGGC TGTTTATTTT GTAACTGGCA AATTAGGCTC TGGAAAGACG CTCGTTAGCG 4801 TTGGTAAGAT TCAGGATAAA ATTGTAGCTG GGTGCAAAAT AGCAACTAAT CTTGATTTAA 4861 GGCTTCAAAA CCTCCCGCAA GTCGGGAGGT TCGCTAAAAC GCCTCGCGTT CTTAGAATAC 4921 CGGATAAGCC TTCTATATCT GATTTGCTTG CTATTGGGCG CGGTAATGAT TCCTACGATG 4981 AAAATAAAAA CGGCTTGCTT GTTCTCGATG AGTGCGGTAC TTGGTTTAAT ACCCGTTCTT 30 5041 GGAATGATAA GGAAAGACAG CCGATTATTG ATTGGTTTCT ACATGCTCGT AAATTAGGAT 5101 GGGATATTAT TTTTCTTGTT CAGGACTTAT CTATTGTTGA TAAACAGGCG CGTTCTGCAT 5161 TAGCTGAACA TGTTGTTTAT TGTCGTCGTC TGGACAGAAT TACTTTACCT TTTGTCGGTA 5221 CTTTATATTC TCTTATTACT GGCTCGAAAA TGCCTCTGCC TAAATTACAT GTTGGCGTTG 5281 TTAAATATGG CGATTCTCAA TTAAGCCCTA CTGTTGAGCG TTGGCTTTAT ACTGGTAAGA 35 5341 ATTTGTATAA CGCATATGAT ACTAAACAGG CTTTTTCTAG TAATTATGAT TCCGGTGTTT 5401 ATTCTTATTT AACGCCTTAT TTATCACACG GTCGGTATTT CAAACCATTA AATTTAGGTC 5461 AGAAGATGAA ATTAACTAAA ATATATTTGA AAAAGTTTTC TCGCGTTCTT TGTCTTGCGA 5521 TTGGATTTGC ATCAGCATTT ACATATAGTT ATATAACCCA ACCTAAGCCG GAGGTTAAAA 5581 AGGTAGTCTC TCAGACCTAT GATTTTGATA AATTCACTAT TGACTCTTCT CAGCGTCTTA 40 5641 ATCTAAGCTA TCGCTATGTT TTCAAGGATT CTAAGGGAAA ATTAATTAAT AGCGACGATT 5701 TACAGAAGCA AGGTTATTCA CTCACATATA TTGATTTATG TACTGTTTCC ATTAAAAAAG 5761 GTAATTCAAA TGAAATTGTT AAATGTAATT AATTTTGTTT TCTTGATGTT TGTTTCATCA 5821 TCTTCTTTTG CTCAGGTAAT TGAAATGAAT AATTCGCCTC TGCGCGATTT TGTAACTTGG 58B1 TATTCAAAGC AATCAGGCGA ATCCGTTATT GTTTCTCCCG ATGTAAAAGG TACTGTTACT 45 5941 GTATATTCAT CTGACGTTAA ACCTGAAAAT CTACGCAATT TCTTTATTTC TGTTTTACGT 6001 GCTAATAATT TTGATATGGT TGGTTCAATT CCTTCCATAA TTCAGAAGTA TAATCCAAAC 6061 AATCAGGATT ATATTGATGA ATTGCCATCA TCTGATAATC AGGAATATGA TGATAATTCC 6121 GCTCCTTCTG GTGGTTTCTT TGTTCCGCAA AATGATAATG TTACTCAAAC TTTTAAAATT 6181 AATAACGTTC GGGCAAAGGA TTTAATACGA GTTGTCGAAT TGTTTGTAAA GTCTAATACT 50 6241 TCTAAATCCT CAAATGTATT ATCTATTGAC GGCTCTAATC TATTAGTTGT TTCTGCACCT 6301 AAAGATATTT TAGATAACCT TCCTCAATTC CTTTCTACTG TTGATTTGCC AACTGACCAG 6361 ATATTGATTG AGGGTTTGAT ATTTGAGGTT CAGCAAGGTG ATGCTTTAGA TTTTTCATTT 6421 GCTGCTGGCT CTCAGCGTGG CACTGTTGCA GGCGGTGTTA ATACTGACCG CCTCACCTCT 6481 GTTTTATCTT CTGCTGGTGG TTCGTTCGGT ATTTTTAATG GCGATGTTTT AGGGCTATCA 55 6541 GTTCGCGCAT TAAAGACTAA TAGCCATTCA AAAATATTGT CTGTGCCACG TATTCTTACG 6601 CTTTCAGGTC AGAAGGGTTC TATCTCTGTT GGCCAGAATG TCCCTTTTAT TACTGGTCGT 6661 GTGACTGGTG AATCTGCCAA TGTAAATAAT CCATTTCAGA CGATTGAGCG TCAAAATGTA 6721 GGTATTTCCA TGAGCGTTTT TCCTGTTGCA ATGGCTGGCG GTAATATTGT TCTGGATATT - 164 6781 ACCAGCAAGG CCGATAGTTT GAGTTCTTCT ACTCAGGCAA GTGATGTTAT TACTAATCAA 6841 AGAAGTATTG CTACAACGGT TAATTTGCGT GATGGACAGA CTCTTTTACT CGGTGGCCTC 6901 ACTGATTATA AAAACACTTC TCAAGATTCT GGCGTACCGT TCCTGTCTAA AATCCCTTTA 6961 ATCGGCCTCC TGTTTAGCTC CCGCTCTGAT TCCAACGAGG AAAGCACGTT ATACGTGCTC 5 7021 GTCAAAGCAA CCATAGTACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 7081 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT 7141 CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC 7201 TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTTGGGTGA 7261 TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC LO 7321 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGG 7381 CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGAA CCACCATCAA ACAGGATTTT 7441 CGCCTGCTGG GGCAAACCAG CGTGGACCGC TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG 7501 AAGGGCAATC AGCTGTTGCC CGTCTCACTG GTGAAAAGAA AAACCACCCT GGATCCAAGC 7561 TTGCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA L5 7621 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT 7681 GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC TTTTTTGCGG 7741 CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA GATGCTGAAG 7801 ATCAGTTGGG CGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG 7861 AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTC 20 7921 ATACACTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGG GCGCGGTATT 7981 CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA 8041 CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG GCCAACTTAC 8101 TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGGATC 8161 ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC 25 8221 GTGACACCAC GATGCCTGTA GCAATGCCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC 8281 TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT AAAGTTGCAG 8341 GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG 8401 GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG CCCTCCCGTA 8461 TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG 30 8521 CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT TACTCATATA 8581 TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG AAGATCCTTT 8641 TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGT ACGTAAGACC 8701 CCCAAGCTTG TCGACTGAAT GGCGAATGGC GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG 8761 TGCCGGAAAG CTGGCTGGAG TGCGATCTTC CTGAGGCCGA TACTGTCGTC GTCCCCTCAA 35 8821 ACTGGCAGAT GCACGGTTAC GATGCGCCCA TCTACACCAA CGTAACCTAT CCCATTACGG 8881 TCAATCCGCC GTTTGTTCCC ACGGAGAATC CGACGGGTTG TTACTCGCTC ACATTTAATG 8941 TTGATGAAAG CTGGCTACAG GAAGGCCAGA CGCGAATTAT TTTTGATGGC GTTCCTATTG 9001 GTTAAAAAAT GAGCTGATTT AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGTT 9061 TACAATTTAA ATATTTGCTT ATACAATCTT CCTGTTTTTG GGGCTTTTCT GATTATCAAC 40 9121 CGGGGTACAT ATGATTGACA TGCTAGTTTT ACGATTACCG TTCATCGATT CTCTTGTTTG 9181 CTCCAGACTC TCAGGCAATG ACCTGATAGC CTTTGTAGAT CTCTCAAAAA TAGCTACCCT 9241 CTCCGGCATG AATTTATCAG CTAGAACGGT TGAATATCAT ATTGATGGTG ATTTGACTGT 9301 CTCCGGCCTT TCTCACCCTT TTGAATCTTT ACCTACACAT TACTCAGGCA TTGCATTTAA 9361 AATATATGAG GGTTCTAAAA ATTTTTATCC TTGCGTTGAA ATAAAGGCTT CTCCCGCAAA 45 9421 AGTATTACAG GGTCATAATG TTTTTGGTAC AACCGATTTA GCTTTATGCT CTGAGGCTTT 9481 ATTGCTTAAT TTTGCTAATT CTTTGCCTTG CCTGTATGAT TTATTGGATG TT 165 0 C

E-E

0 0 E-0 E0 ( 0 E E-0 E- E E- &- 00 < CDU )( E- (Du L' HE- E UE L E- -E- 0 0 L) 0 0( (20 <D C E-L) (D D 0 ~ 0 UD E- F 0 0 0 <D LD u 0 0 u 0 E-D 0 L F. < -00 0 D. E- u0 0) 0 ID- F. UE- E-.- 0 u 4)) < . E -H E- E- ~ 0 E- II ) 0 0 u E- -0 L9I 0 0 AI E, Af In _0 .4 10 L 44 m ~ E- 4~J E4 L4 0 <D > V0 4-4 C-QI $4 (n -<2 -i I-4 C14') 4 ) I C:-- 41s- U)/ 0- OQ J-- _4 .- U u~L 04. (N U < *40 0 0 O m) 0 0 Z 0 0 U)~ II I li =L4 :1 -4 - ~ ..14 0 '00 (I0 L00 00 0 (0 :3 : 3a E- N4= = x(D tA t II-) C3 LOI C r-l -4 (N 166 0) an u Cu u uON0%000o0 ON m ) CP 0' <0'0< O~ ON0 O 0 C)C)C)C))cc I- E-o E-- < 0 -.: 4 oC) E ' U'00 0 u 0 'ON 0 'E-i mt~ L) < 9 E-E- E- E- E- E ON~ '0 mE' (31 m nC% Na i N0 o o 00'm C'm'm O I- - - P F u 0 u 0E. C <u < ON u 0 m m O ON < E E- E- E- E- E- E-0' % C ult 0' 0 u 0 u0 M E u 000Nm000' -E-E E4E-E 0A al 01010 O C C E- E- E E- E-- M (A m 0 0 1 g On0 Nu E C &0 &. - f- 0 cuE- E. I-. u 0 ON m %ai0 On - ml 0% m u E Cu~ ~~~~ - uZ Z C u o u uc E- < EOO OO E- E ) 0 0 167 0 00 0 cc0 u a.CL 00 0. a.0 0 0 0 0 cc L U 0 U Lj-) -4 - 168 Table 25: h3401-h2 captured Via CJ with BsmAI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 S A Q D I Q M T Q S P A T L S aGT GCA Caa gac atc cag atg acc cag tct cca gcc acc ctg tct 5 ! ApaLl... a gcc acc ! L25,L6,L20,L2,L16,A1I Extender.................................Bridge... 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 !V S P G E R A T L S C R A S Q 10 gtg tct cca ggg gaa agg gcc acc ctc tcc tgc agg gcc agt cag 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 !S V S N N L A W Y Q Q K P G Q agt gtt agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag 15 !46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 !V P R L L I Y G A S T R A T D gtt ccc agg ctc ctc atc tat ggt gca tcc acc agg gcc act gat 20 !61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 !I P A R F S G S G S G T D F T atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 25 !L T I S R L E P E D F A V Y Y ctc acc atc agc aga ctg gag cct gaa gat ttt gca gtg tat tac 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 C Q R Y G S S P G W T F G Q G 30 tgt cag cgg tat ggt agc tca ccg ggg tgg acg ttc ggc caa ggg 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 T K V E I K R T V A A P S V F acc aag gtg gaa atc aaa cga act gtg gct gca cca tct gtc ttc 35 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 I F P P S D E Q L K S G T A S atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct 40 ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 !V V C L L N N F Y P R E A K V gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 45 Q W K V D N A L Q S G N S Q E cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 !S V T E Q D S K D S T Y S L S 50 agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc - 169 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 !S T L T L S K A D Y E K H K V agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc 5 196 197 198 199 200 201202 203 204 205 206 207 208 209 210 !Y A C E V T H Q G L S S P V T tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cct gtc aca !211 212 213 214 215 216 217 218 219 220 221 222 223 10 !K S F N K G E C K G E F A aag agc ttc aac aaa gga gag tgt aag ggc gaa ttc gc.....

- 170 Table 26: h3401-d8 KAPPA captured with CJ and BsmAI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 S A Q D I Q M T Q S P A T L S 5 aGT GCA Caa gac ate cag atg acc cag tct cct gcc acc ctg tct ApaL I...Extender.........................a gcc acc ! L25,L6,L20,L2,L16,AII A GCC ACC CTG TCT! L2 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 LO V S P G E R A T L S C R A S Q gtg tct cca ggt gaa aga gcc acc ctc tcc tgc agg gcc agt cag GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L2 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 L5 !N L L S N L A W Y Q Q K P G Q aat ctt ctc agc aac tta gcc tgg tac cag cag aaa cct ggc cag 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 !A P R L L I Y G A S T G A I G ?0 get ccc agg ctc ctc atc tat ggt gct tcc acc ggg gcc att ggt 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 !IPA RFSGSG SGTEFT atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gag ttc act ?5 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 L T I S S L Q S E D F A V Y F ctc acc atc agc agc ctg cag tct gaa gat ttt gca gtg tat ttc 30 ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 !C Q Q Y G T S P P T F G G G T tgt cag cag tat ggt acc tca ccg ccc act ttc ggc gga ggg acc 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 35 !K V E I K R T V A A P S V F I aag gtg gag atc aaa cga act gtg gct gca cca tct gtc tic ate 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 !F P P S D E Q L K S G T A S V 10 tic ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 !V C P L N N F Y P R E A K V Q gtg tgc ccg ctg aat aac tic tat ccc aga gag gcc aaa gta cag 15 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 !W K V D N A L Q S G N S Q E S tgg aag gtg gat aac gcc etc caa tcg ggt aac tcc cag gag agt 50 ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 !V T E Q D N K D S T Y S L S S gtc aca gag cag gac aac aag gac agc acc tac age etc age agc - 171 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 T L T L S K V D Y E K H E V Y acc ctg acg ctg agc aaa gta gac tac gag aaa cac gaa gtc tac 5 ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 !A C E VT H Q G L S S P VT K gcc tgc gaa gtc acc cat cag ggc ctt agc tcg ccc gtc acg aag 211 212 213 214 215 216 217 218 219 220 221 222 223 10 !S F N R G E C K K E F V agc ttc aac agg gga gag tgt aag aaa gaa ttc gtt t -172 Table 27: V3-23 VH framework with variegated codons shown 17 1819 2021 22 5 5'-ctg tet gas cG GCC cap ec CCC atg gcc 29 3-gac aga at gc cgg gtc ggc cgg tac cgg Scab......Sfil1..... NgoM1 ... Ncol.... 10 FR I(DP47T3-23)-------- 23 24 25 26 27 28 29 30 gaalgttlCAAlTTGlttalgagltctggtj 53 15 cIjcaalgtilaaclaat~ctclagalccal I MfelI * 31 323334 3536 3738 39 40 41424344 45 20 GG L VQP GG SL RL SC A Iggclggtlcttlgttica~lct~itttttacgtlctttcttgclgctI 98 Iccgl ccagaa~a Igtcjgga Ices Icca Isga I aa tgca Igas isga IacgIcgs I Sites to be varied-> 25 -FRI-------->I ... CDRI......... -R * 46 4748 49 5051 52 53 5455 56 575859 60 zectlTCCIGGAIttclactlttcltct~tCGITACIGctiatgltctltnnpnttlcgCI 143 l cgalagglcctlaagltgalaaglaglagclatglcgaltacaga*cclcaalgcgI 30 1 BspEI I I BsiWII IBst.XI. Sites to bevaries-> 4 __FR2- - - >I ..CDR2 .... 61 62 63 646566 67 6869 7071 72 7374 75 35 Q QA PG K GL EW VS A IS ICAalgcticcTIG~tlaaagltlp~aelteitlttctlgctjaktcltctlggtI 188 *Igtt Icga Igga Icca ItttIcca IaacIctcIaccI caIgatagagocca ... BstXI I 40 *00 .. CDR2................................ I-FR3 * 76 7778 7980 81 82 83 8485 8687 8889 90 ItctlggtlggclagtjactltacltatlnctleacltccIlnttlaelcgcttcI 233 4 5 ! agajccalccglcatgalatglatalcgalctgjagglcaalttticcalgcgjaagI * 91 92 9394 9596 97 9899100 101 102 103104 105 T IS RD NS KN T LY LQM 50 lactlatclTCTlAGAlgaclaacltctlaaglaatlactlctcltaclttglcagatgI 278 * tgaltaglagaitctlctglttglagalttclttaltgagagatgaacgttacI I Xbal I -FR3---->I 55 ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 N S LR AE DTA V YY CA K laacaaClTTAlA~pejpctlneilgsclaCTACAI~tctatattgclgctlaaaI 323 Ittgltcgiaatitcclcgalctcictgltg*gtlcalatglatalacgcgattt - 173 J AMih I I PstlI ..... CDR3.......... * 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 5 D Y E GTGY AF D IW GQ G IgacltatlgaalggtiactiggtltatlzctittcleaCIATAITG!I!2tcaggtI 368 Ictglatajcttlcca~gaiccaatatgalaagctgltatlacciccaigtticcSI I Ndel I 10 -- - ---- R----- > 136 137 138 139 140 141 142 TM VT VS S lactqatGIGTCIACCigtcltctlagt- 389 ItgaltacicagItgglcaglagaltca 15 1 IBstE11 I * 143 144 145 146 147 148 149 150 151 152 A S T K G P S V F P gcc tcc acc aaG GGC CCa tcg GTC TTC ccc-3' 419 20 cgg agg tppe tic Cep cet apc cap, aaee S. IBsp 1201. Bbs1 ... (2/2) Apal.... (SFPRMET) 5'-ctg tct gaa cG GCC cag ccG-3' (TOPFR I A) 5'-ctg tct gaa cG GCC cag ccG GCC atg, gcc 2 5 gaalgttICAAITTGittalgagltctlggtl. Iggclggtfcttigttlcaglcctlggtlggtltctitla-3' (BOTFRI B) 3'-caalgtclggalccalccalagajaatlgcalgaalagalacglcga IcgaiagglcctIaagjtgaIaag-5' ! bottom strand (BOTFR2) 3'-accicaalgcgl 30 Igttlega Iggal ccalttt Icca Isaaclctc aIaa lags J-5' ! bottom strand (BOTFR3) 3'- alcgalctglagglcaaltttlccalgcglaag Itgaltaglagaltctlctglttglagalttclttaltgalgaglatglaaclgtcltacl jttgltcglaatjtcclcgalctctctgltga.5' (F06) 5'-gCITTAIAGgigctigagigaciaCIlGCAIGtcltacltatltgcjgctlaaal 35 IgacltalgaalggtjactlggtltatlgcllttclgaClATAITGglggtlc-3' (I3OTFR4) 3'-cgalaaglctgitataccccaigttlccai ligaltacicagltgglcaglagaitca cgg sgg tgg tte ccg ggt age cag sag ggg-S'! bottom strand (BOTPRCPRIM) 3'-gg tic ccg ggt agc cag 2ag ggg-5' 40 CDRI diversity (ON-vgC I) 5'-Ig-ctITCClGGAlttelactlttcltctl< I>ITACI< I>Iatcl<1 >4 CDRI ............ 6859 4 5 1t~~tcCCa1c~c1G3 !<I> stands for an equimolar mix of {ADEFGI-IKLMvNPQRSTVWY); no C (this is not a sequence) 50 !CDR2 diversity (ON-vgC2) 5'-ggtlttgfgagltgglgttltctl<2>latci<2>1<3>1 CDR2....... Itctggtlggcl< 1>Iactl< I>Itatlgctlgacitcclgttlaaalgg-3' 55 ! CDR2 ............................... <1> is an equimolar mixture of (ADEFGHIKLMNPQRSTVWY); no C <2> is an equimolar mixture of (YRWVGS); no ACDEFHIKLMNPQT - 174 <3> is an equimolar mixture of {PS); no ACDEFGHIKLMNQRTVWY 175 u -u u<00<U 000(3< ~ < <3 (3 0 U 0 <(:)<Uuooo-<O~o(3 U< <

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N > ~ fa ~ (N1 0a (N...- (rLa 1,~ > oo 4 o0 eq 00 0 N - ce 04 rn u CD to C ca f omccc c 4ma 00 'C4 e 01 O 1Z2 0 0( co toc Do caC oZc C60 cccD f - Q c c c C14 c- C4U co -- m, a:, m( ( t C IO - (N -n 00i< w' CD Q' W%.0 Go CDU C)~ cc N U C1 M VN %0 Z r ; Dz 0 40 t 0' 4 cc T - a, IT 0 0 t o c. 0 le r.020 m C4 W - 00 u 4 = I 00 N 00 co 03N ( a c w Z; rn goC 00 en 00 00 00 vN 00 00 to 00 CI N 00a 2 C 00 C4 C4 * 0 4If r- LC 0 C0 2 !, 189 r- 0 v ca on c 00 0 f~ u 00 r~ U 00 m0 r 0 ~ U 0 e~ r- 00 be C eJZ0 7 fn mJ0 -au -, w c rq d 00 - r ~ C t- to r4. co t t- *y~ oo o m - CL UO N00 0 - -f 00 10 0 00 0 00 d. I fn > o-nz m 00 -- - a ~~~~~ 00 wl co~ 0- 0' 0 . r-0 00 in o 1 '<D u0 00 : 0 ~C to 000 *r C _ o 00 C, ~ 00o0 ~c0 /u %2 (N Z E O 0 Zo ~ ) wC 0 V a< .QV 03~0 -00 CO on 00 - m 0 0 n o 0 n 00 (n~ Un 0o a :; C4 en ;- 00 V 00 00 - 2Q '0o C4r 00 -r o C 0 00 o U0 C4 co ~ 00 00 to %0 00 .0 CO - O ~ 0~ m a 0 "W' u Do a 00 < acC~ v-w 0 < Go CY CO 0_ U - -e to e4> tj> c'a 0 Wu C4' 0 0a *D 00 _,, cc0 co0- 0 CD0 en l C)r L C) r4 fn 00 en a.> L) rn L 2! * v.w < 190 -e 0%, 0% 0% en 00 C en-bf 00 en00 '0 m0 r- 00 C7, Go C 4oa e n e~~ ~ ~ 00 -I o Vwl a )% C- a 1 t- 0 " -V u % 00 W) wl00o u ~ Uo U - < 00 ba w C40 W~ - -0 0 u ~ %n e- -j 0 Go >C C-4§ -- u LL Go~.n-v - 03 qf ~ ~ O (7 0-t 0000 ~ U 0 C o% < 0 Z 0Z a wC a~0 (c) 00Urv< o 00 0 < 00 en) 000 u -W > 000. %0~00 0W 00 m 0%0 'i - Z r- 2 L'- = t- N' 0 cu' 00 r- 00 0% C* - C13 00 "o. L) 'n 7 -o 00 '0 %n cisu 00 < U %0 l0 -n 00 00 00 N'O 000 ' In 0 %n %n 0 C 191 L)0 < u ED I- 0 co) 00 00.0u -u V0 . ( ooo 000 to~- moo u0 u l 0 0 0 ' 00 00 0 0 U M co -0 04 - 00 u Go~ M d 00 -. 000c0 00 C to ~ ~ 0 00wu u 0 *0o co0 00 00-co c to 00 DO CM 0j 00 0 0 u (00v On u - u 0c 0 0 0 .0 a 0 E0 00 00 I 0~~ 0( an 00 Q, 00 to0, u (J00 am co (' - 0 .5 00 wo o 000 0u 00 00"U o o o w00 00 co 00 00 QQ ' uu cu C4 UM 00 mu m u00 00 z 10 1 E .g u - U UM 00~ m u 0 00 o % "J 0 00 I00z 000M 000 00 00.0 0 00 Zil0 u - n - mU 0 U '- 00 (0D 00 00 0 -r a = -- O CL ~ 0 ~~~~~>. 000 O 0 * '- - 1 rJ LC)L C) LOl - 192 Table 30: Oligonucleotides used to clone CDRI/2 diversity All sequences are 5' to 3'. l) ON_CDI Bsp, 30 bases 5 AccTcAcTggcTTccgg A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 T T c A c T T T c T c T LO 192021222324252627282930 2) ON_Brl2, 42 bases AgAAAcccAcTccAAAcc L 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 TTTAccAggAgcTTggcg 192021222324252627282930313233343536 ?0 A A c c c A 37 38 39 40 4142 3) ONCD2Xba, 51 bases 25 ggAAggcAgTgATcTAgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 gATAgTgA AgcgAccTTT 192021222324252627282930313233343536 30 A Acgg AgTcAgcATA 37 38 39 40 4142 43 44 45 46 47 48 49 50 51 35 4) ONBotXba, 23 bases ggAAggcAgTgATcTAgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 10 g A T A g 192021 2223 - 193 Table 31: Bridge/Extender Oligonucleotides ON LamlaB7(rc) .........................GTGCTGACTCAGCCACCCTC. 20 ON^Lam2aB7(rc) ........................GCCCTGACTCAGCCTGCCTC. 20 ON Lam3lB7(rc) ....................... GAGCTGACTCAGG.ACCCTGC 20 5 ON-Lam3rB7(rc) ........................GAGCTGACTCAGCCACCCTC. 20 ON LamHflcBrg (rc) CCTCGACAGCGAAGTGCACAGAGCGTCTTGACTCAGCC....... .. 38 ON LamHflcExt CCTCGACAGCGAAGTGCACAGAGCGTCTTG ............... ... 30 ON LamH f2b25rg (rc) CCTCGACAGCGAAGTGCACAGAGCGCTTTGACTCAGCC....... .. 38 ON LamHf2b2Ext CCTCGACAGCGAAGTGCACAGAGCGCTTTG............... ... 30 10 ONLamH f2dBrg (rc) CCTCGACAGCTAAGTGCACAGAGCGCTTTGACTCAGCC....... .. 38 ON LamH f2dExt CCTCGACAGCGAAGTGCACAGAGCGCTTTG ............... . 30 ON LamHf3lBrg (rc) CCTCGACAGCGAAGTGCACAGAGCGAATTGACTCAGCC....... .. 38 ON LamHIf3lExt CCTCGACAGCGAAGTGCACAGAGCGAATTG ............... . 30 ON LamH f3rBrg (rc) CCTCGACAGCGAAGTGCACAGTACGAATTGACTCAGCC....... .. 38 15 ON LamH f3rExt CCTCGACAGCGAAGTGCACAGTACGAATTG............... . 30 ON lamPlePCR CCTCGACAGCGAAGTGCACAG. ............................. 21 Consensus - 194 Table 32: Oligonucleotides used to make SSDNA locally double-stranded Adapters (8) H43HF3.1?02#1 5'-cc gtg tat tac tgt gcg aga g-3' 5 H43.77.97.1-03#2 5'-ct gtg tat tac tgt gcg aga g-3' H43.77.97.323#22 5'-cc gta tat tac tgt geg aTa g-3' H43.77.97.330#23 5'-ct gtg tat tac tgt gcg aaa g-3' H43.77.97.439#44 5'-ct gtg tat tac tgt gcg aga e-3' H43.77.97.551#48 5'-cc !atg tat tac tgt gcg aga g-3' - 195 Table 33: Bridge/extender pairs Bridges (2) H43.XABr1 5 5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg ctgaggacaCTGCAGtctactattgtgcgaga-3' H43.XABr2 5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg 10 ctgaggacaCTGCAGtctactattgtgcgaaa-3' Extender H43.XAExt 5'ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTcATcTgcAAgTAgAgAgTATTcTTAg 15 AgTTgTcTcTAgATcAcTAcAcc-3' - 196 Table 34: PCR primers Primers H43.XAPCR2 gactgggTgTAgTgATcTAg 5 Hucmnest cttttctttgttgccgttggggtg - 197 Table 35: PCR program for amplification of heavy chain CDR3 DNA 95 degrees C 5 minutes 95 degrees C 20 seconds 5 60 degrees C 30 seconds repeat 20x 72 degrees C 1 minute 72 degrees C 7 minutes 4 degrees C hold Reaqents (100 ul reaction): 10 Template Sul ligation mix 10x PCR buffer 1x Taq 5U dNTPs 200 uM each MgCl2 2mM 15 H43.XAPCR2-biotin 400 nM Hucmnest 200 nM - 198 Table 36: Annotated sequence of CJR DY3F7(CJR-A05) 10251 bases Non-cutters 5 BclI Tgatca BsiWI Cgtacg BssSI Cacgag !BstZl7I GTAtac BtrI CACgtg EcoRV GATatc !FseI GGCCGGcc HpaI GTTaac Mlul Acgcgt !PmeI GTTTaaac PmlI CACgtg PpuMI RGgwccy !RsrII CGgwccg SapI GCTCTTC SexAI Accwggt .0 !SgfI GCGATcgc SgrAI CRccggyg SphI GCATGc !StuI AGGcct XmaI Cccggg cutters L5 Enzymes that cut from 1 to 4 times and other features !End of genes II and X 829 !Start gene V 843 !BsrGI Tgtaca 1 1021 20 !BspMI Nnnnnnnnngcaggt 3 1104 5997 9183 !-"- ACCTGCNNNNn 1 2281 !End of gene V 1106 !Start gene VII 1108 !BsaBI GATNNnnatc 2 1149 3967 ?5 !Start gene IX 1208 !End gene VII 1211 !SnaBI TACgta 2 1268 7133 !BspHI Tcatga 3 1299 6085 7093 !Start gene VIII 1301 30 !End gene IX 1304 !End gene VIII 1522 !Start gene III 1578 !EagI Cggccg 2 1630 8905 !XbaI Tctaga 2 1643 8436 35 !KasI Ggcgcc 4 1650 8724 9039 9120 !BsmI GAATGCN 2 1769 9065 !BseRI GAGGAGNNNNNNNNNN 2 2031 8516 !-"- NNnnnnnnnnctcctc 2 7603 8623 !AlwNI CAGNNNctg 3 2210 8072 8182 40 !BspDI ATcgat 2 2520 9883 !NdeI CAtatg 3 2716 3796 9847 !End gene III 2846 !Start gene VI 2848 !AfeI AGCgct 1 3032 45 !End gene VI 3187 !Start gene I 3189 !EarI CTCTTCNnnn 2 4067 9274 !-"- Nnnnngaagag 2 6126 8953 !PacI TTAATtaa 1 4125 50 !Start gene IV 4213 !End gene I 4235 !BsmFI Nnnnnnnnnnnnnnngtccc 2 5068 9515 !MscI TGGcca 3 5073 7597 9160 !PsiI TTAtaa 2 5349 5837 55 !End gene IV 5493 !Start ori 5494 !NgoMIV Gccggc 3 5606 8213 9315 !BanII GRGCYc 4 5636 8080 8606 8889 !DraIII CACNNNgtg 1 5709 60 !DrdI GACNNNNnngtc 1 5752 !AvaI Cycgrg 2 5818 7240 - 199 !PvuII CAGctg 1 5953 !BsmBI CGTCTCNnnnn 3 5964 8585 9271 !End ori region 5993 !BamHI Ggatcc 1 5994 5 !HindIII Aagctt 3 6000 7147 7384 !BciVI GTATCCNNNNNN 1 6077 !Start bla 6138 !Eco57I CTGAAG 2 6238 7716 !SpeI Actagt 1 6257 10 !BcgI gcannnnnntcg 1 6398 !ScaI AGTact 1 6442 !PvuI CGATcg 1 6553 !FspI TGCgca 1 6700 !BglI GCCNNNNnggc 3 6801 8208 8976 15 !BsaI GGTCTCNnnnn 1 6853 !AhdI GACNNNnngtc 1 6920 !Eaml105I GACNNNnngtc 1 6920 !End bla 6998 !AccI GTmkac 2 7153 8048 20 !HincII GTYrac 1 7153 !SalI Gtcgac 1 7153 !XhoI Ctcgag 1 7240 !Start PlacZ region 7246 !End PlacZ region 7381 25 !PflMI CCANNNNntgg 1 7382 !RBS1 7405 !start M13-iii signal seq for LC 7418 !ApaLI Gtgcac 1 7470 !end M13-iii signal seq 7471 30 !Start light chain kappa L20:JK1 7472 !PflFI GACNnngtc 3 7489 8705 9099 !SbfI CCTGCAgg 1 7542 !PstI CTGCAg 1 7543 !KpnI GGTACc 1 7581 35 !XcmI CCANNNNNnnnntgg 2 7585 9215 !NsiI ATGCAt 2 7626 9503 !BsgI ctgcac 1 7809 !BbsI gtcttc 2 7820 8616 !BlpI GCtnagc 1 8017 40 !EspI GCtnagc 1 8017 !EcoOl09I RGgnccy 2 8073 8605 !Ecl136I GAGctc 1 8080 !SacI GAGCTc 1 8060 !End light chain 8122 45 !AscI GGcgcgcc 1 8126 !BssHII Gcgcgc 1 8127 !RBS2 8147 !SfiI GGCCNNNNnggcc 1 8207 !NcoI Ccatgg 1 8218 50 !Start 3-23, FR1 8226 !MfeI Caattg 1 8232 !BspEI Tccgga 1 8298 !Start CDR1 8316 !Statt FR2 8331 55 !BstXI CCANNNNNntgg 2 8339 8812 !EcoNI CCTNNnnnagg 2 8346 8675 !Start FR3 8373 !XbaI Tctaga 2 8436 1643 !AflII Cttaag 1 8480 60 !Start CDR3 8520 !AatII GACGTc 1 8556 - 200 !Start FR4 8562 !PshAI GACNNnngtc 2 8573 9231 !BstEII Ggtnacc 1 8579 !Start CHI 8595 5 !ApaI GGGCCc 1 8606 !Bspl20I Gggccc 1 8606 !PspOMI Gggccc 1 8606 !AgeI Accggt 1 8699 !Bsu36I CCtnagg 2 8770 9509 .0 !End of CHI 8903 !NotI GCggccgc 1 8904 !Start His6 tag 8913 !Start cMyc tag 8931 !Amber codon 8982 5 !NheI Gctagc 1 8985 !Start M13 III Domain 3 8997 !NruI TCGcga 1 9106 !BstBI TTcgaa 1 9197 !EcoRI Gaattc 1 9200 .0 !XcmI CCANNNNNnnnntgg 1 9215 !BstAPI GCANNNNntgc 1 9337 !SacII CCGCgg 1 9365 !End IIIstump anchor 9455 !AvrII Cctagg 1 9462 .5 !trp terminator 9470 !SwaI ATTTaaat 1 9784 !Start gene II 9850 !BglII Agatct 1 9936 10 1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc gene ii continued 49 cca aat gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta 97 tct aat ggt caa act aaa tct act cgt tcg cag aat tgg gaa tca act 145 gtt aTa tgg aat gaa act tcc aga cac cgt act tta gtt gca tat tta 15 193 aaa cat gtt gag cta cag caT TaT att cag caa tta agc tct aag cca 241 tcc gca aaa atg acc tct tat caa aag gag caa tta aag gta ctc tct 289 aat cct gac ctg ttg gag ttt gct tcc ggt ctg gtt cgc ttt gaa gct 337 cga att aaa acg cga tat ttg aag tct ttc ggg ctt cct ctt aat ctt 385 ttt gat gca atc cgc ttt gct tct gac tat aat agt cag ggt aaa gac t0 433 ctg att ttt gat tta tgg tca ttc tcg ttt tct gaa ctg ttt aaa gca 481 ttt gag ggg gat tca ATG aat att tat gac gat tcc gca gta ttg gac Start gene x, ii continues 529 gct atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct 577 ttt gca aaa gcc tct cgc tat ttt ggt ttt tat cgt cgt ctg gta aac 15 625 gag ggt tat gat agt gtt gct ctt act atg cct cgt aat tcc ttt tgg 673 cgt tat gta tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg 721 atg aat ctt tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att 769 aac gta gat ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt 817 ctt aaa atc gca TAA 0 ! End X & II 832 ggtaattca ca ! M1 ES Q10 T15 843 ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt >5 !Start gene V ! S17 S20 P25 E30 891 tct ggt gtt tct cgt cag ggc aag cct tat tca ctg aat gag cag ctt 0 V35 E40 V45 939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ctt gtc aag att act - 201 D50 A55 L60 987 ctt gat gaa ggt cag cca gcc tat gcg cct ggt cTG TAC Azc gtt cat BsrGI... 5 L65 V70 S75 R80 1035 ctg tcc tct ttc aaa gtt ggt cag ttc ggt tcc ctt atg att gac cgt P85 K87 end of V 1083 ctg cgc ctc gtt ccg gct aag TAA C 10 1108 ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg Start gene VII 1150 ata caa atc tcc gtt gta ctt tgt ttc gcg ctt ggt ata atc 15 VII and IX overlap. ..... S2 V3 L4 VS S10 1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttT gcc tct ttc gtt End VII 20 Istart IX L13 W15 G20 T25 E29 1242 tta ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta atg gaa 1293 act tcc tc 25 ! .... stop of IX, IX and VIII overlap by four bases 1301 ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct acc ctc Start signal sequence of viii. 30 1349 gtt ccg atg ctg tct ttc gct gct gag ggt gac gat ccc gca aaa gcg mature VIII --- > 1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat gcg 1445 tgg gcg atg gtt gtt gtc att 1466 gtc ggc gca act atc ggt atc aag ctg ttt aag 35 bases 1499-1539 are probable promoter for iii 1499 aaa ttc acc tcg aaa gca ! 1515 ........... -35 40 1517 agc tga taaaccgat acaattaaag gctccttttg ..... -10 ... 1552 gagccttttt ttt GGAGAt ttt ! S.D. uppercase, there may be 9 Ts 45 <------ III signal sequence ----------------------------- > M K K L L F A I P L V V P F 1574 caac GTG aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc ! 1620 Y S G A A E S H L D G A 50 1620 tat tct ggc gCG GCC Gaa tca caT CTA GAc ggc gcc EagI.... XbaI.... Domain 1 ----------------------------------------------------------- A E T V E S C L A 55 1656 gct gaa act gtt gaa agt tgt tta gca K S H T E I S F T N V W K D D K T 1683 aaA Tcc cat aca gaa aat tca ttt aCT AAC GTC TGG AAA GAC GAC AAA ACt 60 L D R Y A N Y E G S L W N A T G V 1734 tta gat cgt tac gct aac tat gag ggC tgt ctg tgG AAT GCt aca ggc gtt - 202 BsmI.... V V C T G D E T Q C Y G T W V P I 1785 gta gtt tgt act ggt GAC GAA ACT CAG TGT TAC GGT ACA TGG GTT cct att 5 G L A I P E N 1836 ggg ctt gct atc cct gaa aat Li linker ----------------------------------- LO E G G G S E G G G S 1857 gag ggt ggt.ggc tct gag ggt ggc ggt tct E G G G S E G G G T 1887 gag ggt ggc ggt tct gag ggt ggc ggt act 15 Domain 2 ------------------------------------ 1917 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat atc aac 1968 cct ctc gac ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct 2019 aat cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag aat 20 BseRI.. 2070 aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc act 2118 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct 2166 gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt aaa ttC AGA AlwNI 25 2214 GAC TGc gct ttc cat tct ggc ttt aat gaG gat TTa ttT gtt tgt gaa AlwNI 2262 tat caa ggc caa tcg tct gac ctg cct caa cct cct gtc aat gct 2307 ggc ggc ggc tct 30 ! start L2 ------------------------------------------------------------ 2319 ggt ggt ggt tct 2331 ggt ggc ggc tct 2343 gag ggt ggt ggc tct gag gga ggc ggt tcc 2373 ggt ggt ggc tct ggt end L2 35 Many published sequences of M13-derived phage have a longer linker than shown here by repeats of the EGGGS motif two more times. Domain 3 ------------------------------------------------------------- 40 ! S G D F D Y E K M A N A N K G A 2388 tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct M T E N A D E N A L Q S D A K G 2436 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc 45 K L D S V A T D Y G A A M D G F 2484 aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc I G D V S G L A N G N G A T G D 50 2532 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat F A G S N S Q M A Q V G D G D N 2580 ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat 55 S P L M N N F R Q Y L P S L P Q 2628 tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa S V E C R P F V F G A G K P Y E 2676 tcg gtt gaa tgt cgc cct ttt gtc ttt Ggc gct ggt aaa cca tat gaa 60 * F S I D C D K I N L F R - 203 2724 ttt tct att gat tgt gac aaa ata aac tta ttc cgt End Domain 3 G V F A F L L Y V A T F M Y V F140 5 2760 ggt gte ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt start transmembrane segment S T F A N I L 2808 tct acg ttt gct aac ata ctg 10 R N K E S 2829 cgt aat aag gag tct TAA ! stop of iii Intracellular anchor. 15 Ml P2 V L L5 G I P L L10 L R F L G15 2847 tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc ctc ggt Start VI 2894 ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag 20 2942 ggc ttc ggt aag ata get att get att tea ttg ttt ett get ctt att 2990 att ggg ctt aac tea att ctt gtg ggt tat ctc tct gat att agc get 3038 caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att ctc ccg tct 3086 aat gcg ctt ccc tgt ttt tat gtt att ctc tct gta aag get gct att 3134 ttc att ttt gac gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat 25 Ml A2 V3 F5 L10 G13 3182 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga end VI Start gene I 30 K T L V S V G K I Q D K I V A 3228 aag acg ctc gtt agc gtt ggt aag att cag gat aaa att gta gct G C K I A T N L D L R L Q N L 3273 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac ctc 35 P Q V G R F A K T P R V L R I 3318 ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata P D K P S I S D L L A I G R G 40 3363 ceg gat aag cet tet ata tot gat ttg ctt get att ggg cgc ggt N D S Y D E N K N G L L V L D 3408 aat gat tcc tac gat gaa aat aaa aac ggc ttg ctt gtt ctc gat 45 E C G T W F N T R S W N D K E 3453 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa R Q P I I D W F L H A R K L G 3498 aga cag ccg att att gat tgg ttt cta cat gct cgt aaa tta gga 50 W D I I F L V Q D L S I V D K 3543 tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa Q A R S A L A E H V V Y C R R 55 3588 cag gcg cgt tct gca tta get gaa cat gtt gtt tat tgt cgt cgt L D R I T L P F V G T L Y S L 3633 ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt 60 I T G S K M P L P K L H V G V 3678 att act ggc tcg aaa atg cct ctg cct aaa tta cat gtt ggc gtt - 204 V K Y G D S Q L S P T V E R W 3723 gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg 5 L Y T G K N L Y N A Y D T K Q 3768 ctt tat act ggt aag aat ttg tat aac gca tat gat act aaa cag A F S S N Y D S G V Y S Y L T 3813 gct ttt tct agt aat tat gat tcc ggt gtt tat tct tat tta acg 10 P Y L S H G R Y F K P L N L G 3858 cct tat tta tca cac ggt cgg tat ttc aaa cca tta aat tta ggt Q K M K L T K I Y L K K F S R 15 3903 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc V L C L A I G F A S A F T Y S 3948 gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt aca tat agt 20 Y I T Q P K P E V K K V V S Q 3993 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag ! T Y D F D K F T I D S S Q R L 4038 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt 25 N L S Y R Y V F K D S K G K L 4083 aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA PacI 30 I N S D D L Q K Q G Y S L T Y 4128 ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat ! PacI ! iI D L C T V S I K K G N S N E 35 ! iv Ml K 4173 att gat tta tgt act gtt tcc att aaa aaa ggt aat tca aAT Gaa Start IV ! i I V K C N .End of I 40 iv L3 L N5 V 17 N F V10 4218 att gtt aaa tgt aat TAA T TTT GTT IV continued..... 4243 ttc ttg atg ttt gtt tca tca tct tct ttt gct cag gta att gaa atg 4291 aat aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca 45 4339 ggc gaa tcc gtt att gtt tct ccc gat gta aaa ggt act gtt act gta 4387 tat tca tct gac gtt aaa cct gaa aat cta cgc aat ttc ttt att tct 4435 gtt tta cgt gcA aat aat ttt gat atg gtA ggt tcT aAC cct tcc atT 4483 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca 4531 tca tct gat aat cag gaa tat gat gat aat tcc gct cct tct ggt ggt 50 4579 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat 4627 aac gtt cgg gca aag gat tta ata cga gtt gtc gaa ttg ttt gta aag 4675 tct aat act tct aaa tcc tca aat gta tta tct att gac ggc tct aat 4723 cta tta gtt gtt agt gcT cct aaa gat att tta gat aac ctt cct caa 4771 ttc ctt tcA act gtt gat ttg cca act gac cag ata ttg att gag ggt 55 4819 ttg ata ttt gag gtt cag caa ggt gat gct tta gat ttt tca ttt gct 4867 gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc 4915 ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat 4963 ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat 5011 tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag aag 60 5059 ggt tct atc tct gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg MscI....

- 205 5107 act ggt gaa tct gcc aat gta aat aat cca ttt cag acg att gag cgt 5155 caa aat gta ggt att tcc atg agc gtt ttt cct gtt gca atg gct ggc 5203 ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct 5251 tct act cag gca agt gat gtt att act aat caa aga agt att gct aca 5 5299 acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggc ctc act 5347 gat tat aaa aac act tct caG gat tct ggc gta ccg ttc ctg tct aaa 5395 atc cct tta atc ggc ctc ctg ttt agc tcc cgc tct gat tcT aac gag 5443 gaa agc acg tta tac gtg ctc gtc aaa gca acc ata gta cgc gcc ctg 5491 TAG cggcgcatt 10 End IV 5503 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 5563 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcGCCGGCt ttccccgtca NgoMI. 5623 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 15 5683 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt DraIII.... 5743 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac DrdI.......... 5803 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 20 5863 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5923 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga PvuII. BsmBI. 5983 aaaaccaccc tGGATCC AAGCTT BamHI HindIII (1/2) 25 Insert carrying bla gene 6006 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc 6043 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct BciVI 6103 gataaatgct tcaataatat tgaaaaAGGA AGAgt 30 RBS.?... Start bla gene 6138 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt 6189 tgc ctt cct gtt ttt gct cac cca gaa acg ctg qtg aaa gta aaa gat gct 6240 gaa gat cag ttg ggC gcA CTA GTg ggt tac atc gaa ctg gat ctc aac agc 35 Spel.... ApaLI & BssSI Removed 6291 ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc 6342 act ttt aaa gtt ctg cta tgt GGC GcG Gta tta tcc cgt att gac gcc ggg 6393 caa gaG CAA CTC GGT CGc cgC ATA cAC tat tct cag aat gac ttg gtt gAG 40 BcgI............ ScaI 6444 TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa ScaI. 6495 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta ctt 6546 ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg 45 PvuI.... 6597 ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc 6648 ata cca aac gac gag cgt gac acc acg atg cct gta gca atg Gca aca acg 6699 tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct tcc cgg caa caa ! FspI.... 50 6750 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg 6801 GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt ! Bg1I .......... 6852 gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt 55 BsaI.... 6903 atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat AhdI........... 6954 aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg TAA ctgt stop 60 7003 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 7063 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt - 206 7123 cgttccactg tacgtaagac cccc 7147 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct ! HindIl SalI.. (2/2) HincII 5 7183 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt Start of Fab-display cassette, the Fab DSR-A05, selected for binding to a protein antigen. 10 7233 CCTGAcG CTCGAG xBsu36I XhoI.. PlacZ promoter is in the following block 15 7246 cgcaacgc aattaatgtg agttagctca 7274 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 7324 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7374 tgattacgCC AagcttTGGa gccttttttt tggagatttt caac PflMI....... 20 Hind3. (there are 3) Gene iii signal sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M K K L L F A I P L V V P F Y 7418 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat 25 ! 16 17 18 Start light chain (L20:JK1) ! S H S A Q D I Q M T Q S P A 7463 tct cac aGT GCA Caa gac atc caq atq acc caq tct cca gcc ApaLI... 30 Sequence supplied by extender............ T L S L 7505 acc ctg tct ttg 35 S P G E R A T L S C R A S Q G 7517 tct cca ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag Ggt ! V S S Y L A W Y Q Q K P G Q A 7562 gtt age agc tac tta gcc tgg tac cag cag aaa cct ggc cag get 40 P R L L I Y D A S S R A T G I 7607 ccc agg ctc ctc atc tat gAt gca tcc aAc agg gcc act ggc atc ! P A R F S G S G P G T D F T L 45 7652 cca gCc agg ttc agt ggc agt ggg Cct ggg aca gac ttc act etc T I S S L E P E D F A V Y Y C 7697 acc atc agc agC ctA gag cct gaa gat ttt gca gtT tat tac tgt 50 Q Q R S W H P W T F G Q G T R 7242 cag cag CGt aAc tgg cat ccg tgg ACG TTC GGC CAA GGG ACC AAG V E I K R T V A A P S V F I F 7787 gtg gaa atc aaa cga act gtg gCT GCA Cca tct gtc ttc atc ttc 55 BsgI.... P P S D E Q L K S G T A S V V 7832 ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg 60 C L L N N F Y P R E A K V Q W 7877 tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg - 207 K V D N A L Q S G N S Q E S V 7922 aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc 5 T E R D S K D S T Y S L S S T 7967 aca gag cgg gac agc aag gac agc acc tac agc ctc agc agc acc L T L S K A D Y E K H K V Y A 8012 ctg acG CTG AGC aaa gca gac tac gag aaa cac aaa gtc tac gcc 10 EspI..... C E V T H Q G L S S P V T K S 8057 tgc gaa gtc acc cat cag ggc ctG AGC TCg ccc gtc aca aag agc SacI.... 15 F N R G E C 8102 ttc aac agg gga gag tgt taa taa 8126 GGCGCG CCaattctat ttcaaGGAGA cagtcata 20 AscI..... RBS2. PeiB signal sequence------(22 codons)-----> 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M K Y L L P T A A A G L L L L 25 8160 atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc ... PelB signal------------> Start VH, FRI-----------------> 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A A Q P A M A E V Q L L E S G 30 8205 gcG GCC cag ccG GCC atg gcc gaa gtt CAA TTG tta gag tct ggt SfiI............. MfeI... NcoI.... 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 35 G G L V Q P G G S L R L S C A 8250 ggc ggt ctt gtt cag cct ggt ggt tct tta cgt ctt tct tgc gct ... FRI--------------------> CDR1--------------> FR2--------> 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 40 A S G F T F S T Y E M R W V R 8295 gct TCC GGA ttc act ttc tct act tac gag atg cgt tgg gtt cgC BspEI.. BstXI... FR2---------------------------------------> CDR2 ---------- > 45 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Q A P G K G L E W V S Y I A P 8340 CAa gct ccT GGt aaa ggt ttg gag tgg gtt tct tat atc gct cct BstXI................ 50 ... CDR2----------------------------------------------> FR3----> 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 S G G D T A Y A D S V K G R F 8385 tct ggt ggc gat act gct tat gct gac tcc gtt aaa ggt cgc ttc 55 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 T I S R D N S K N T L Y L Q M 8430 act atc TCT AGA qac aac tct aaq aat act ctc tac ttq caq atq XbaI... Supplied by extender------------------------------ 60 - ----------------------------------------- FR3--------------> - 208 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 N S L R A E D T A V Y Y C A R 8475 aac aqC TTA AGg qct gaq gac act qca qtc tac tat tgt gcg agg AflII... 5 from extender---------------------------------> ! CDR3---------------------------------------------------> FR4--> 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 R L D G Y I S Y Y Y G M D V W LO 8520 agg ctc gat ggc tat att tcc tac tac tac ggt atg GAC GTC tgg AatII.. 136 137 138 139 140 141 142 143 144 145 G Q G T T V T V .S S L5 8565 ggc caa ggg acc acG GTC ACC gtc tca agc BstEII... CH1 of IgGl----------> A S T K G P S V F P L A P S S 20 8595 gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc K S T S G G T A A L G C L V K 8640 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag 25 D Y F P E P V T V S W N S G A 8685 gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc L T S G V H T F P A V L Q S S 8730 ctg acc agc ggc gtc cac acc ttc ccg gct gtc cta cag tCC TCA 30 Bsu36I.. G L Y S L S S V V T V P S S S 8775 GGa ctc tac tcc ctc agc agc gta gtq acc gtq ccc tcc agc agc Bsu36I.... 35 L G T Q T Y I C N V N H K P S 8820 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc N T K V D K K V E P K S C A A 40 8865 aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt GCG GCC NotI...... A H H H H H H G A A E Q K L I 8910 GCa cat cat cat cac cat cac ggg gcc gca gaa caa aaa ctc atc 45 ..NotI.... H6 tag................. Myc-Tag........................ S E E D L N G A A q A S S A 8955 tca gaa gag gat ctg aat ggg gcc gca tag GCT AGC tct gct Myc-Tag.................... ... NheI... 50 Amber III'stump Domain 3 of III ------------------------------------------------------ 55 S G D F D Y E K M A N A N K G A 8997 agt ggc gac ttc gac tac gag aaa atg gct aat gcc aac aaa GGC GCC tcc t t t t t a g a c t t g g t !W.T. KasI...(2/4) 60 M T E N A D E N A L Q S D A K G - 209 9045 atG ACT GAG AAC GCT GAC GAG aat gct ttg caa agc gat gcc aag ggt c a t c t a c g c a g tct c t a c !W.T. K L D S V A T D Y G A A I D G F 5 9093 aag tta gac agc gTC GCG Acc gac tat GGC GCC gcc ATC GAc ggc ttt a c t t tct t t t c t t t t t c !W.T. NruI .... KasI... (3/4) I G D V S G L A N G N G A T G D 10 9141 atc ggc gat gtc agt ggt tTG GCC Aac ggc aac gga gcc acc gga gac t t c t tcc c c t t t t t t t t t t !W.T. MscI....:3/3) F A G S N S Q M A Q V G D G D N 15 9189 ttc GCA GGT tcG AAT TCt cag atg gcC CAG GTT GGA GAT GGg gac aac t t c t c a t a c t c t t t !W.T. BspMI.. (2/2) XcmI................ EcoRI... 20 S P L M N N F R Q Y L P S L P Q 9237 agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg cag tca t t a t t c c t a t t a t c c t a !W.T. S V E C R P F V F S A G K P Y E 25 9285 agt gtc gag tgc cgt cca ttc gtt ttc tct gcc ggc aag cct tac gag tcg t a t c t t c t agc t t a a t a !W.T. ! F S I D C D K I N L F R 9333 ttc aGC Atc gac TGC gat aag atc aat ctt ttC CGC 30 t tct t t t c a a c t a c t !W.T. BstAPI........ SacII... End Domain 3 G V F A F L L Y V A T F M Y V F 35 9369 GGc gtt ttc gct ttc ttg cta tac gtc gct act ttc atg tac gtt ttc t c t g t c t t a t t c c t t a t !W.T. start transmembrane segment S T F A N I L R N K E S 40 9417 aGC ACT TTC GCC AAT ATT TTA Cgc aac aaa gaa agc tct g t t c a c g t t g g tct !W.T. Intracellular anchor. 45 9453 tag tga tct CCT AGG AvrII.. 9468 aag ccc gcc taa tga gcg ggc ttt ttt ttt ct ggt I Trp terminator 50 End Fab cassette 9503 ATGCAT CCTGAGG ccgat actgtcgtcg tcccctcaaa ctggcagatg NsiI.. Bsu36I.(3/3) 55 9551 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg 9611 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc 9671 tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg 9731 agctgattta acaaaaattt aaTgcgaatt ttaacaaaat attaacgttt acaATTTAAA SwaI... 60 9791 Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat 9850 ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc - 210 Start gene II 9901 tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata BglII... 9952 gct acc ctc tcc ggc atT aat tta tca gct aga acg gtt gaa tat cat-att 5 10003 gat ggt gat ttg act gtc tcc ggc ctt tct cac cet ttt gaa tct tta cct 10054 aca cat tac tca ggc att gca ttt aaa ata tat gag gg- tct aaa aat ttt 10105 tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt cat 10156 aat gtt ttt ggt aca acc gat tta get tta tgc tct gag get tta ttg ctt 10207 aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt 10 gene II continues ------------------------ End of Table ------------------------------- - 211 Table 37: DNA seq of w.t. M13 gene iii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 fM K K L L F A I P L V V P F Y 5 1579 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat Signal sequence............................................ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 S H S A E T V E S C L A K P H 10 1624 tct cac tcc gct gaa act gtt gaa agt tgt tta gca aaa ccc cat Signal sequence>. Domain 1-------------------------------------- 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 T E N S F T N V W K D D K T L 15 1669 aca gaa aat tca ttt act aac gtc tgg aaa gac gac aaa act tta Domain 1-------------------------------------------------- 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 D R Y A N Y E G C L W N A T G 20 1714 gat cgt tac gct aac tat gag ggt tgt ctg tgG AAT GCt aca ggc BsmI.... ! Domain 1-------------------------------------------------- ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 25 V V V C T G D E T Q C Y G T W 1759 gtt gta gtt tgt act ggt gac gaa act cag tgt tac ggt aca tgg ! Domain 1-------------------------------------------------- ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 30 V P I G L A I P E N E G G G S 1804 gtt cct att ggg ctt gct atc cct gaa aat gag ggt ggt ggc tct ! Domain 1-------------------------------> Linker 1---------- ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 35 E G G G S E G G G S E G G G T 1849 gag ggt ggc ggt tct gag ggt ggc ggt tct gag ggt ggc ggt act Linker 1--------------------------------------------------> 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 40 K P P E Y G D T P I P G Y T Y 1894 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat Domain 2-------------------------------------------------- 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 45 I N P L D G T Y P P G T E Q N 1939 atc aac cct ctc gac ggc act taT CCG CCt ggt act gag caa aac Ecil.... Domain 2-------------------------------------------------- 50 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 P A N P N P S L E E S Q P L N 1984 ccc gct aat cct aat cct tct ctt GAG GAG tct cag cct ctt aat BseRI.. Domain 2--------------------------------------------------- 55 ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 ! T F M F Q N N R F R N R Q G A 2029 act ttc atg ttt cag aat aat agg ttc cga aat agg cag ggg gca ! Domain 2-------------------------------------------------- 60 ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 - 212 L T V Y T G T V T Q G T D P V 2074 tta act gtt tat acg ggc act gtt act caa ggc act gac ccc gtt Domain 2------------------------------------------------- 5 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 K T Y Y Q Y T P V S S K A M Y 2119 aaa act tat tac cag tac act cct gta tca tca aaa gcc atg tat Domain 2-------------------------------------------------- LO 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 D A Y W N G K F R D C A F H S 2164 gac gct tac tgg aac ggt aaa ttC AGa gaC TGc gct ttc cat tct AlwNI....... Domain 2-------------------------------------------------- L5 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 G F N E D P F V C E Y Q G Q S 2209 ggc ttt aat gaG GAT CCa ttc gtt tgt gaa tat caa ggc caa tcg BamHI... Domain 2-------------------------------------------------- 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 S D L P Q P P V N A G G G S G 2254 tct gac ctg cct caa cct cct gtc aat gct ggc ggc ggc tct ggt 25 Domain 2------------------------------> Linker 2---------- 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 G G S G G G S E G G G S E G G 2299 ggt ggt tct ggt ggc ggc tct gag ggt ggt ggc tct gag ggt ggc 30 Linker 2-------------------------------------------------- 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 G S E G G G S E G G G S G G G 2344 ggt tct gag ggt ggc ggc tct gag gga ggc ggt tcc ggt ggt ggc 35 Linker 2--------------------------------------------------- 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 S G S G D F D Y E K M A N A N 2389 tct ggt tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat 40 !Linker 2> Domain 3------------------------------------------- 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 K G A M T E N A D E N A L Q S 2434 aag ggg gct atg acc gaa aat gcc gat gaa aac gcg cta cag tct 45 Domain 3--------------------------------------------------- 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 D A K G K L D S V A T D Y G A 2479 gac gct aaa ggc aaa ctt gat tct gtc gct act gat tac ggt gct 50 Domain 3--------------------------------------------------- 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 ! A I D G F I G D V S G L A N G 2524 gct atc gat ggt ttc att ggt gac gtt tcc ggc ctt gct aat ggt 55 Domain 3--------------------------------------------------- 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 N G A T G D F A G S N S Q M A 2569 aat ggt gct act ggt gat ttt gct ggc tct aat tcc caa atg gct 60 Domain 3---------------------------------------------------- - 213 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 Q V G D G D N S P L M N N F R 2614 caa gtc ggt gac ggt gat aat tca cct tta atg aat aat ttc cgt Domain 3-------------------------------------------------- 5 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 Q Y L P S L P Q S V E C R P F 2659 caa tat tta cct tcc ctc cct caa tcg gtt gaa tgt cgc cct ttt Domain 3--------------------------------------------------- 10 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 V F S A G K P Y E F F I D C D 2704 gtc ttt agc gct ggt aaa cca tat gaa ttt tct att gat tgt gac ! Domain 3-------------------------------------------------- 15 ! 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 ! K I N L F R G V F A F L L Y V 2749 aaa ata aac tta ttc cgt ggt gtc ttt gcg ttt ctt tta tat gtt ! Domain 3--------------> Transmembrane segment------------- 20 ! 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 ! A T F M Y V F S T F A N I L R 2794 gcc acc ttt atg tat gta ttt tct acg ttt gct aac ata ctg cgt ! Transmembrane segment---------------------------------> ICA- 25 ! 421 422 423 424 425 ! N K E S 2839 aat aag gag tct taa ! 2853 ! ICA ----------- > ICA = intracellular anchor 30 ------------------ End of Table ----------------------------------------- - 214 Table 38: Whole mature III anchor M13-III derived anchor with recoded DNA 1 2 3 5 A A A 1 GCG gcc gca NotI...... 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1.0 H H H H H H G A A E Q K L I 10 cat cat.cat cac cat cac ggg gcc gca gaa caa aaa ctc atc 18 19 20 21 22 23 24 25 26 27 28 29 S E E D L N G A A . A S L5 52 tca gaa gag gat ctg aat ggg gcc gca Tag GCT AGC NheI... 30 31 32 33 34 35 36 37 38 39 D I N D D R M A S T 88 GAT ATC aac pat gat cot atq gct tct act (ONG37bot) [RC) 5'-c aac gat gat cot ato gcG CAt Gct gcc gag aca g-3' EcoRV.. Enterokinase cleavage site. 25 Start mature III (recoded) Domain 1 ---- > 40 41 42 43 A E T V 118 IgcCjgaGlacAlgtCI t a t t ! W.T. 30 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 E S C L A K P H T E N S F T N 130 Igaa|TCCitgCICTGIGCCIAaGIccTicaClacTIgaGlaatIAGTIttClaCA|AatI agt t t a a a c t a a tca t t c W.T. 35 MscI.... 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 V W K D D K T L D R Y A N Y E 175 IgtglTGGlaaGIgaTigaTIaaGlacCICtTIgATICGAITaTlgcClaaTitaCIgaAI 40 c a c c a t t a t c t c t g ! W.T. BspDI... 74 75 76 77 78 79 80 81 82 83 84 85 86 82 88 G C L W N A T G V V V C T G D 45 220 IggCltgClTtAltgglaatIgcCIACCIGGCIGtCIgtTIgtCITGCIACGIggClgaTI t t c g t a t a t t t t c ! W.T. SgrAI...... BsgI.... 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 50 E T Q C Y G T W V P I G L A I 265 IgaGlacAlcaAltgCltaTIggCIACGITGglgtGlccGlatAIgGCITTAIGCClatAI a t g t c t a t t t g c t t c! W.T. PmlI.... BlpI..... 55 Domain 1-----> Linker 1----------------> 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 P E N E G G G S E G G G S E G 310 IccGIgaGlaaCIgaAlggCIggCIggTIAGClgaAlggCIggTIggCIAGClgaAlggCI t a t g t t c tct g t c t tct g t W.T. 60 Linker 1----------------------> Domain 2---------------> - 215 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 G G S E G G G T K P p E Y G D 355 IggTIGGAITCCIgaAlggAlggTlggAlacClaaGIccGIccGigaAltaTIggCIgaCI c t t g t c t t a t t g c t t W.T. 5 BamHI..(2/2) 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 T P I P G Y T Y I N P L D G T 400 lacTIccGlatAICCTIGGTItaClacCItaClatTlaaTIccGITtAlgaTIggAlacCI 10 a t t g c t t t c c t c c c c t !W.T. SexAI.... 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 y P P G T E Q N P A N P N P S 15 445 taC IccTI ccGIggCI acCIgaAIcaGIaaTIccTI gcCIaaCIccGI aaCI ccAIAGCI T G t t t g a c c t t t t t tct ! W.T. Hindill... 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 20 L E E S Q P L N T F M F Q N N 490 ITTAIgaAlgaAIAGCIcaAlccGITtAlaaClacCIttTlatglttCIcaAlaaClaaCI ! c t G G tct g t c t t t c t g t t! W.T. HindIII. 25 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 R F R N R Q G A L T V Y T G T 535 |CgTIttT|AgGlaaCICgTIcaAlgGTIGCTICtTlacCigTGITACIAcTIggAlacCI a g c c a t a g g g a t a t t t g c t W.T. HgiAI... BsrGI... 30 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 V T Q G T D P V K T Y Y Q Y T 580 IgtClacCIcaGIGGTIACCIgaTIccTIgtClaaGlacCItaCitaTIcaAltaTlacCI t t a c t c c t a t t c g c t ! W.T. 35 KpnI... 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 P V S S K A M Y D A Y W N G K 625 IccGIgtCITCGIAGtlaaGlgcTlatgltaCIgaTlgcCItaTItgglaaTiggClaaGI 40 t a a tca a c t c t c c t a W.T. BsaI.... XhoI.... 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 45 F R D C A F H S G F N E D P F 670 IttTICgTIgaTItgTlgcCIttTIcaCIAGCIggTIttClaaClgaalgacICCtlttTI C A a C c t c t tct c t t G T a c W.T. 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 50 V C E Y Q G Q S S D L P Q P P 715 IgtCItgClgaGItaCIcaGIggTIcaGIAGTIAGCIgaTITtAIccGtcaGIccAICCGI t t a t a c a tcg tct c c g t a t t !W.T. DrdI..... AgeI..... 55 Domain 2--------> Linker 2---------------------> 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 V N A G G G S G G G S G G G S 760 IGTTIAAClgcGIggTIggTIggTIAGCIggCIggAIggCIAGClggCIggT | ggTIAGCI C t t c c c tct t t t tct t c c tct ! W.T. 60 AgeI..... HpaI...

- 216 HincII. Linker 2----------------------------------------------> Domain 3--> 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 5 E G G G S E G G G S G G G S G 805 IgaAlggCIggAIggTIAGCIgaAlggAIggTiggCIAGClggAIggCIggTIAGClggCI g t t c tct g t c t tct g t c tct t !W.T. ------------- Domain 3--------------------> LO 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 S G D F D Y E K M A N A N K G 850 AGflggClgaclttclgacltaclgaglaaalatglgctlaatIgcclaaclaaalGGCI tcc t t t t t a g a c t t g g! W.T. KasI.... L5 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 A M T E N A D E N A L Q S D A 895 IGCClatglactigaglaaclgct igaclgaG|AATIGCAlctglcaaIagtigatIgCCI t c a t c t a c g a g tct c t ! W.T. KasI.... BsmI .... Styl... 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 K G K L D S V A T D Y G A A I 940 |AAGIGGt laaglttalgacIagc|gTCIGCcIAcalgacItat |ggTIGCtlgcclatc 5 a c a c t t tct t t t c t !W.T. StyI...... PflFI...... 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 D G F I G D V S G L A N G N G 30 985 igaclggcItttiatclggclgatIgtclagtIggtIctglgctIaaclggclaaclggaI t t c t t c t tcc c c t t t t t! W.T. 344 345 346 347 348 349 350 351 352 353 A T G D F A G S N S 35 1030 IgcclaccIggaIgacIttclGCAIGGTitcG|AATITCtI t t t t t t c t c !W.T. BstBI... EcoRI... BspMI.. 10 354 355 356 357 358 359 360 361 362 363 Q M A Q V G D G D N 1060 cag atg gcC CAG GTT GGA GAT GGg gac aac a t a c t c t t t !W.T. X5 xcmI................ 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 S P L M N N F R Q Y L P S L P Q 1090 agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg cag 50 tca t t a t t c c t a t t a t c c t a W.T. 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 ! S V E C R P F V F S A G K P Y E 1138 agt gtc gag tgc cgt cca ttc gtt ttc tct gcc ggc aag cct tac gag 55 tog t a t c t t c t agc t t a a t a W.T. ! Domain 3--------------------------------------> 396 397 398 399 400 401 402 403 404 405 406 407 ! F S I D C D K I N L F R 1186 t.: aGC Atc gac TGC gat aag atc aat ctt ttC CGC St tct t t t c a a c t a t - 217 BstAPI........ SacII... transmembrane segment-------------> 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 5 G V F A F L L Y V A T F M Y V F 1222 GGc gtt ttc gct ttc ttg cta tac gtc gct act ttc atg tac gtt ttc t c t g t c t t a t t c c t t a t !W.T. 424 425 426 427 428 429 430 431 432 433 434 435 10 S T F A. N I L R N K E S 1270 aGC ACT TTC GCC AAT ATT TTA Cgc aac aaa gaa agc tct g t t c a c g t t g q tct ! W.T. Intracellular anchor. 15 1306 tag tga tct CCT AGG AvrII.. 1321 aag ccc gcc taa tga gcg ggc ttt ttt ttt ct ggt 20 1 Trp terminator End Fab cassette ---------------------------- End of Table ------------------------- - 218 Table 39: ONs to make deletions in III ONs for use with NheI N 5 (ONG29bot) 5'-c gTT gAT ATc gcT Agc cTA Tgc-3' 22 this is the reverse complement of 5'-gca tag gct agc gat atc aac g-3' NheI... scab......... (ON_G104top) 5'-glatalggclttalgcTlaGCiccgigaglaaclgaalgg-3' 10 30 Scab..........NheI... 104 105 106 107 108 (ONG236top) 5--cittticaciagclggtttclGCTIAGClgacicctItttIgtcltgc-3' 37 NheI ... 236 237 238 239 240 15 (ONG236tCS) 5'-citttIcaclagcIggtlttclGCTIAGCgacIcctItttIgtclAgc NheI... 236 237 238 239 240 gagitacIcagjggtc-3' 50 ! ONs for use with SphI G CAT Gc 20 (ONX37bot) 5'-gAc TgT cTc ggc Agc ATg cgc cAT Acg ATc ATc gTT g-3' 37 N D D R M A H A (ONX37bot)=[RC) 5'-c aac gat gat cgt atg gcG CAt Gct gcc gag aca gtc-3' SphI ....Scab........... 25 (ON_X104top) 5'-glgtG ccglatalggclttGlCATIGCalccglgaglaaclgaalgg-3' 36 Scab................SphI .... 104 105 106 107 108 (ONX236top) 5'-clttticaclagclggtttGICaTlgCalgaclcctltttlgtcltgc-3' 37 30 SphI .... 236 237 238 239 240 (ONX236tCS) 5'-clttticaclagclggtlttGlCaTlgCalgaclcctltttlgtclAgc NheI ... 236 237 238 239 240 gagltaclcaglggtlc-3' 50 - 219 Table 40: Phage titers and enrichments of a selections with a DY3F31-based human Fab library Input (total cfu) Output (total cfu) Output/input ratio Ri-ox selected on 4,5 x 102 3,4 x 10' 7,5 x 10 5 phOx-BSA * R2-Strep selected 9,2 x 1012 3 x l0 3,3 x 10 on Strep-beads - 220 Table 41: Frequency of ELISA positives in DY3F31-based Fab libraries Anti-M13 HRP 9E10/RAM- Anti-CK/CL HRP Gar-HRP R2-ox (with IPTG induction) 18/44 10/44 10/44 R2-ox (without IPTG) 13/44 ND ND 5 R3-strep (with IPTG) 39/44 38/44 36/44 R3-strep (without IPTG) 33/44 ND ND

Claims (27)

1. A method for cleaving a nucleic acid at a desired location, the method comprising the steps of: (i) contacting a single-stranded nucleic acid with a single stranded oligonucleotide, the single-stranded oligonucleotide being complementary to the single-stranded nucleic acid in the region in which cleavage is desired; wherein the single-stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the locally double-stranded region com prises a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid at the restriction endonuclease recognition, wherein the cleaving comprises contacting a restriction endonuclease to the locally double-stranded region, wherein the restriction endonuclease is specific for the restriction endonuclease recognition site; the contacting and the cleaving steps being performed at a tempera ture wherein the single-stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the remainder of the single-stranded nucleic acid is single-stranded, and wherein the restriction endonuclease is active at the temperature.

2. The method of claim 1, wherein nucleic acid encodes at least a portion of an immunoglobulin.

3. The method of claim 2, wherein the immunoglobulin comprises a Fab or single-chain Fv.

4. The method of claim 2 or claim 3, wherein the immunoglobulin comprises at least a portion of a heavy chain.

5. The method of claim 2 or claim 3, wherein the immunoglobulin comprises at least a portion of FR1. - 222

6. The method of claim 2 or claim 3, wherein the immunoglobulin comprises at least a portion of a light chain.

7. The method of any one of claims 4 to 6, wherein at least a portion of the heavy chain, FR1 or light chain is human.

8. The method of any one of claims 1 to 7, wherein the nucleic acid is at least in part derived from a patient suffering from at least one autoimmune disease or cancer.

9. The method of claim 8, wherein the autoimmune disease is lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospho lipid syndrome or vasculitis.

10. The method of claim 8, wherein the nucleic acid is at least in part isolated from peripheral blood cells, bone marrow cells, spleen cells, or lymph node cells.

11. The method of any one of claims claim 1 to 10, wherein the temperature is selected from: between 45*C and 75*C; between 50'C and 60*C; or between 55*C and 60*C.

12. The method of any one of claims 1 to 11, wherein the length of the single-stranded oligonucleotide is between 17 and 30 bases.

13. The method of claim 12, wherein the length of the single stranded oligonucleotide is between 18 and 24 bases.

14. The method of any one of claims 1 to 13, wherein the restriction endonuclease is selected from the group consisting of: MaeIII, Tsp451, HphI, BsaJI, AluI, BlpI, DdeI, BglII, MslI, BsiEI, EaeI, EagI, HaeIII, Bst4Cl, HpyCH4III, HinfI, MlyI, PleI, MnlI, HpyCH4V, BsmAI, BpmI, XmnI, TaaI, and SacI; or Bst4CI, TaaI, HpyCH4III, BlpI, HpyCH4V, and MslI. - 223

15. The method of claim 2, or any one of claim 3 to 14 when dependent on claim 2, wherein the immunoglobulin comprises synthetic diversity in at least one of CDR1 or CDR2 and/or natural diversity in CDR3.

16. The method of claim 15, wherein the natural diversity in CDR3 is from B cells.

17. The method of any one of claims 1 to 16, wherein the single stranded nucleic acid has been amplified prior to cleavage.

18. The method of any one of claims 1 to 17, wherein the single stranded nucleic acid comprises a synthetic sequence attached to the 5' end and the synthetic sequence is cleaved from the nucleic acid by the method of claim 1.

19. The method of claim 18, wherein the single-stranded nucleic acid has been amplified prior to cleavage with a primer which binds to at least a portion of the synthetic sequence.

20. The method of any one of claims 1 to 19, wherein the single stranded nucleic acid is immobilized.

21. The method of claim 20, wherein the immobilized single-stranded nucleic sequence is cleaved at the restriction endonuclease cleavage site to thereby obtain a released single-stranded nucleic acid sequence.

22. The method of claim 21, wherein the released single-stranded nucleic acid sequence is ligated to an adapter oligonucleotide.

23. The method of claim 22, wherein the adaptor oligonucleotide comprises a double-stranded region comprising a nucleic acid sequence of one or more codons of the amino acid sequence encoded by the released single-stranded nucleic acid sequence.

24. The method of claim 23, wherein the double-stranded region is 12 to 100 bases in length. - 224

25. The method of claim 23, wherein the double-stranded region com prises a restriction endonuclease recognition site.

26. The method of claim 22, wherein the adaptor oligonucleotide comprises a single stranded region complementary to the region of the cleavage in the released single-stranded nucleic acid sequence.

27. The method of claim 26, wherein the single-stranded region is 2 to 15 bases in length. Date: 8 December 2011