CA2360796A1 - Improved helper dependent vector system for gene therapy - Google Patents
Improved helper dependent vector system for gene therapy Download PDFInfo
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- CA2360796A1 CA2360796A1 CA002360796A CA2360796A CA2360796A1 CA 2360796 A1 CA2360796 A1 CA 2360796A1 CA 002360796 A CA002360796 A CA 002360796A CA 2360796 A CA2360796 A CA 2360796A CA 2360796 A1 CA2360796 A1 CA 2360796A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The present invention features helper-dependent adenoviral vector elements, and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors. Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' ITR that confers a selective advantage, and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50 % to about 60 %. The modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus. The E4 non-coding segment and the stuffer region(s) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.
Description
TITLE OF THE INVENTION
IMPROVED HELPER DEPENDENT VECTOR SYSTEM FOR GENE THERAPY
FIELD OF THE INVENTION
This invention relates to adenoviral vectors which are useful for nucleic acid delivery into a cell such as in gene delivery and nucleotide vaccine applications. It also relates to host cells and methods using these vectors.
BACKGROUND OF THE INVENTION
The references cited herein are not admitted to be prior art to the claimed invention.
Adenoviral vectors provide a vehicle for introducing nucleic acids into a cell in vitro and in vivo. Over 100 different serotypes, of which 47 are of human origin, have been reported. (Hierholzer, et al., J. infect. Dis. 159:804-813.
1988.) Adenoviruses type 2 and 5 used to produce adenoviral vectors are well characterized.
A human adenovirus contains a 5' inverted terminal repeat (TTR); a packaging signal; region El made up of the early regions ElA and E1B; region E2;
region E3, region E4; the late regions Ll-L5; and a 3' ITR. Regions E1 and E4 contain regulatory proteins, region E2 encodes proteins required for replication, and the L region encodes for the structural proteins of the virus. The E3 region is dispensable for virus growth in vitro. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) A replicating viral vector based on the adenovirus must contain adenovirus cis elements needed for replication. Packaging also requires cis elements on the vector whereas the necessary proteins can be supplied in traps. In traps supplementation can be brought about by specialized cells and/or additional viruses producing the needed proteins. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) Different types of adenoviral vectors have been developed including those lacking one or more components of the adenoviral genome. An adenoviral vector produced without components of the adenoviral genome provides advantages such as in increasing the amount of foreign nucleic acid that can be included with the adenoviral vector and removing adenoviral genes whose expression can have detrimental effects. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) Adenoviral vectors lacking all viral protein-coding sequences have been developed. These vectors require supplementation of viral regulatory and structural proteins supplied i~2 traps for packaging and rescue. A second adenovirus carrying genes necessary for virus growth can be used to provide in traps the required supplementation of proteins. (Mitani, et al., Proc. Natl. Acad. Sci. USA
92:3854-3858, 1995; Fisher, et al., Virology 217:11-22, 1996; Kochanek, et al., Proc.
Natl.
Acad. Sci. USA 93:5731-5736, 1996; Parks, et al., Proc. Natl. Acad. Sci. USA
93:13565-13570, 1996; Parks, et al., J. Virology 71 (4):3293-3298, 1997; and Schiedner, et al., Nature Genetics 18:180-183, 1998.) SUMMARY OF THE INVENTION
The present invention features helper-dependent adenoviral vector elements, and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors. Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' TTR that confers a selective advantage, and stuffer regions) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. The modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus. The E4 non-coding segment and the stuffer regions) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.
A "helper-dependent adenoviral vector" refers to a viral vector containing the cis elements needed for adenovirus replication and packaging (also referred to herein as viral generation), but lacking the necessary traps elements for adenovirus replication and packaging. The cis elements needed for viral generation is an adenovirus 3' TTR, a packaging signal, and an adenovirus 5' IRT. Traps elements needed for viral generation are the proteins encoded by the El, E2, and E4 and L
adenovirus regions. Preferably, the helper-dependent adenoviral vector lacks nucleic acid encoding for any adenovirus proteins. A helper-dependent adenoviral vector is particularly useful as a vector for delivering genes into cells in vivo.
A "helper virus" refers to a virus expressing one or more proteins needed for viral generation of a helper-dependent adenovirus. Preferably, the helper virus contains the adenovirus 3' IRT, an adenovirus packaging signal, an adenovirus 5' IRT, and encodes for the proteins of the Adenovirus E2 and E4 and L
regions.
Traps elements not provided for by a helper virus may be provided for by other means such as by a cell. For example, a 293 cell can be employed to supply in traszs needed E1 proteins in conjunction with a helper virus not expressing such proteins.
Helper-dependent adenoviral vectors can be produced by co-cultivating with helper viruses in cell lines, wherein the helper virus alone or in combination with the cell line provide the traps functions needed for adenovirus generation. During co-cultivation of a helper-dependent adenoviral vector and a helper virus the two compete for packaging, producing a mixed viral population.
Often, during co-cultivation the helper virus has a selection advantage, such that the mixed population of helper virus and helper-dependent adenoviral vector becomes predominantly helper virus, rather than the desired helper-dependent adenoviral vector. Even when this phenomenon does not occur, the presence of lesser, but significant, amounts of helper viruses still contaminates the helper-dependent adenoviral vector preparation. Therefore, the viral populations may have to be separated physically and/or production of helper particles suppressed for the helper-dependent adenoviral vector to be used. Preferably, the percentage of helper virus in a purified helper-dependent adenoviral vector stock is below about 0.5°70.
Thus, a first aspect of the present invention describes a nucleic acid molecule comprising an excisable low homology packaging signal cassette. The packaging cassette comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence. The modified packaging signal has low homology to a wild-type adenovirus packaging signal.
"Flanked by a recombinase recognition sequence" indicates the presence of a recombinase recognition sequence 3' and 5' of the packaging signal allowing for excision of the flanked nucleic acid containing the packaging signal by a recombinase recognizing the recognition sequence. The recombinase recognition sequence does not need to be immediately 3' or 5' of the modified packaging signal.
Preferably, the 3' and 5' recognition sequence are about 200 by to about 2,000 by apart.
The modified packaging signal has low homology to a wild-type adenovirus packaging signal and still allows for packaging of the helper virus during its separate production. Preferably, the modified packaging signal is less efficient than a wild-type adenovirus packaging signal.
IMPROVED HELPER DEPENDENT VECTOR SYSTEM FOR GENE THERAPY
FIELD OF THE INVENTION
This invention relates to adenoviral vectors which are useful for nucleic acid delivery into a cell such as in gene delivery and nucleotide vaccine applications. It also relates to host cells and methods using these vectors.
BACKGROUND OF THE INVENTION
The references cited herein are not admitted to be prior art to the claimed invention.
Adenoviral vectors provide a vehicle for introducing nucleic acids into a cell in vitro and in vivo. Over 100 different serotypes, of which 47 are of human origin, have been reported. (Hierholzer, et al., J. infect. Dis. 159:804-813.
1988.) Adenoviruses type 2 and 5 used to produce adenoviral vectors are well characterized.
A human adenovirus contains a 5' inverted terminal repeat (TTR); a packaging signal; region El made up of the early regions ElA and E1B; region E2;
region E3, region E4; the late regions Ll-L5; and a 3' ITR. Regions E1 and E4 contain regulatory proteins, region E2 encodes proteins required for replication, and the L region encodes for the structural proteins of the virus. The E3 region is dispensable for virus growth in vitro. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) A replicating viral vector based on the adenovirus must contain adenovirus cis elements needed for replication. Packaging also requires cis elements on the vector whereas the necessary proteins can be supplied in traps. In traps supplementation can be brought about by specialized cells and/or additional viruses producing the needed proteins. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) Different types of adenoviral vectors have been developed including those lacking one or more components of the adenoviral genome. An adenoviral vector produced without components of the adenoviral genome provides advantages such as in increasing the amount of foreign nucleic acid that can be included with the adenoviral vector and removing adenoviral genes whose expression can have detrimental effects. (Hitt, et al., Advances in Pharmacology 40:137-206, 1996.) Adenoviral vectors lacking all viral protein-coding sequences have been developed. These vectors require supplementation of viral regulatory and structural proteins supplied i~2 traps for packaging and rescue. A second adenovirus carrying genes necessary for virus growth can be used to provide in traps the required supplementation of proteins. (Mitani, et al., Proc. Natl. Acad. Sci. USA
92:3854-3858, 1995; Fisher, et al., Virology 217:11-22, 1996; Kochanek, et al., Proc.
Natl.
Acad. Sci. USA 93:5731-5736, 1996; Parks, et al., Proc. Natl. Acad. Sci. USA
93:13565-13570, 1996; Parks, et al., J. Virology 71 (4):3293-3298, 1997; and Schiedner, et al., Nature Genetics 18:180-183, 1998.) SUMMARY OF THE INVENTION
The present invention features helper-dependent adenoviral vector elements, and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors. Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' TTR that confers a selective advantage, and stuffer regions) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. The modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus. The E4 non-coding segment and the stuffer regions) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.
A "helper-dependent adenoviral vector" refers to a viral vector containing the cis elements needed for adenovirus replication and packaging (also referred to herein as viral generation), but lacking the necessary traps elements for adenovirus replication and packaging. The cis elements needed for viral generation is an adenovirus 3' TTR, a packaging signal, and an adenovirus 5' IRT. Traps elements needed for viral generation are the proteins encoded by the El, E2, and E4 and L
adenovirus regions. Preferably, the helper-dependent adenoviral vector lacks nucleic acid encoding for any adenovirus proteins. A helper-dependent adenoviral vector is particularly useful as a vector for delivering genes into cells in vivo.
A "helper virus" refers to a virus expressing one or more proteins needed for viral generation of a helper-dependent adenovirus. Preferably, the helper virus contains the adenovirus 3' IRT, an adenovirus packaging signal, an adenovirus 5' IRT, and encodes for the proteins of the Adenovirus E2 and E4 and L
regions.
Traps elements not provided for by a helper virus may be provided for by other means such as by a cell. For example, a 293 cell can be employed to supply in traszs needed E1 proteins in conjunction with a helper virus not expressing such proteins.
Helper-dependent adenoviral vectors can be produced by co-cultivating with helper viruses in cell lines, wherein the helper virus alone or in combination with the cell line provide the traps functions needed for adenovirus generation. During co-cultivation of a helper-dependent adenoviral vector and a helper virus the two compete for packaging, producing a mixed viral population.
Often, during co-cultivation the helper virus has a selection advantage, such that the mixed population of helper virus and helper-dependent adenoviral vector becomes predominantly helper virus, rather than the desired helper-dependent adenoviral vector. Even when this phenomenon does not occur, the presence of lesser, but significant, amounts of helper viruses still contaminates the helper-dependent adenoviral vector preparation. Therefore, the viral populations may have to be separated physically and/or production of helper particles suppressed for the helper-dependent adenoviral vector to be used. Preferably, the percentage of helper virus in a purified helper-dependent adenoviral vector stock is below about 0.5°70.
Thus, a first aspect of the present invention describes a nucleic acid molecule comprising an excisable low homology packaging signal cassette. The packaging cassette comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence. The modified packaging signal has low homology to a wild-type adenovirus packaging signal.
"Flanked by a recombinase recognition sequence" indicates the presence of a recombinase recognition sequence 3' and 5' of the packaging signal allowing for excision of the flanked nucleic acid containing the packaging signal by a recombinase recognizing the recognition sequence. The recombinase recognition sequence does not need to be immediately 3' or 5' of the modified packaging signal.
Preferably, the 3' and 5' recognition sequence are about 200 by to about 2,000 by apart.
The modified packaging signal has low homology to a wild-type adenovirus packaging signal and still allows for packaging of the helper virus during its separate production. Preferably, the modified packaging signal is less efficient than a wild-type adenovirus packaging signal.
"Low homology" refers to a maximum of about 25 by of contiguous sequence homology (100% sequence identity) between the modified packaging signal and a wild type packaging signal present in an adenovirus, preferably human adenovirus serotype 5. Low homology is determined by aligning the modified packaging signal with wild type packaging signal to obtain a stretch of maximum contiguous homology with sequences present in both the modified and wild type packaging signals. Preferably, contiguous sequence homology is at most 23 bp.
"Less efficient" than a wild-type adenovirus packaging signal indicates that the helper virus has a lower level of packaging when it contains the modified packaging signal than it has when it contains a wild-type packaging signal present in an adenovirus, preferably from a human adenovirus group C serotype, more preferably serotype 5. Preferably, the helper virus can still be produced at yields of at least 300 PFU/cell in cells lacking a compatible recombinase. Efficiency is preferably determined using titration by end point dilution assay described in the Examples below.
Reference to "modified" is not a limitation as to how the packaging signal sequence is produced. Instead, "modified" indicates that the sequence is not a wild-type sequence.
Another aspect of the present invention describes an adenoviral helper virus for production of helper-dependent vectors comprising:
(a) an adenovirus genome having an El region deletion;
(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP
site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal;
and (c) an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.
Another aspect of the present invention describes a helper-dependent adenovirus vector comprising the following elements (in 5' to 3' order): (a) a 5'-inverted terminal repeat sequence; (b) a packaging signal; (c) one or more heterologous gene expression cassettes; (d) an optionally present E4 non-coding segment conferring a selective advantage; and (e) a 3' ITR; wherein (d) and (e) are located in the distal 400 by of the adenovirus genome, and wherein the only adenoviral sequences present in the vector are said 5'-inverted terminal repeat, said 3'-inverted terminal repeat, said packaging signal, and said nonexpressed segment of the E4 region.
The "E4 non-coding segment" refers to the nonexpressed segment of the E4 region co-localized with the E4 promoter. The segment is adjacent to the 3' TTR and, in human adenovirus serotype 5, is about 300 by in length.
Another aspect of the present invention describes a helper-dependent adenovirus vector having a GC content between about 50°70 and about 60%. The vector comprises in a 5' to 3' direction: (a) a 5' ITR; (b) a packaging signal cassette directly joined to the 3' of the 5' TTR; (c) a first stuffer DNA at least about 1 kb; (d) at least one heterologous expression cassette; (e) a second stuffer DNA at least about 1 kb; (f) an optionally present non-coding E4 segment; and (g) a 3' ITR. Element (f) if present is directly joined to element (g). The virus does not encode one or more adenovirus proteins needed for adenovirus generation and is about 28 kb to about 36 kb. Preferably, the virus does not encode any adenovirus proteins.
"Directly joined" indicates the absence of significant intervening sequences (e.g., less than about 150 bp) that interfere with the function of "joined"
groups.
"Stuffer sequences" refer to nucleic acid regions that do not express protein when present in a helper-dependent adenoviral vector. Preferably, such regions are mammalian non-gene regions or introns.
Another aspect of the present invention describes a cell line infected with a helper virus, a helper-dependent viral vector, or both the virus and the vector.
Preferably, the cell line expresses El proteins and a recombinase, and is infected with a helper virus that (1) contains an excisable low homology signal packaging cassette, and (2) does not express El proteins needed for viral generation.
Another aspect of this invention is a method of generating helper-dependent adenoviral gene vectors in a cell line expressing E1 proteins and Cre recombinase comprising:
a) infecting the cell line with a helper-dependent vector comprising: a 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present;
b) infecting the cell line with a helper virus comprising: an adenovirus genome having an E1 region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.
Another aspect of the present invention describes a method of generating helper-dependent adenoviral vectors. The method involves producing a cell comprising: (i) traps functions needed for adenovirus generation and (ii) a helper-dependent adenoviral vector comprising the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, wherein the helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.
The cell is used to generate the helper-dependent vector.
Reference to a cell comprising the traps functions needed for adenovirus generation indicates such functions are present in the cell, but need not be part of the cellular genome. The traps functions can be provided, for example, by a helper virus, the cellular genome, or a combination of both. Another example of a source of traps functions is a plasmid.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples.
The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention.
Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the shuffling of fragments of a stuffer to prevent genomic integration of the expression units.
"Less efficient" than a wild-type adenovirus packaging signal indicates that the helper virus has a lower level of packaging when it contains the modified packaging signal than it has when it contains a wild-type packaging signal present in an adenovirus, preferably from a human adenovirus group C serotype, more preferably serotype 5. Preferably, the helper virus can still be produced at yields of at least 300 PFU/cell in cells lacking a compatible recombinase. Efficiency is preferably determined using titration by end point dilution assay described in the Examples below.
Reference to "modified" is not a limitation as to how the packaging signal sequence is produced. Instead, "modified" indicates that the sequence is not a wild-type sequence.
Another aspect of the present invention describes an adenoviral helper virus for production of helper-dependent vectors comprising:
(a) an adenovirus genome having an El region deletion;
(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP
site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal;
and (c) an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.
Another aspect of the present invention describes a helper-dependent adenovirus vector comprising the following elements (in 5' to 3' order): (a) a 5'-inverted terminal repeat sequence; (b) a packaging signal; (c) one or more heterologous gene expression cassettes; (d) an optionally present E4 non-coding segment conferring a selective advantage; and (e) a 3' ITR; wherein (d) and (e) are located in the distal 400 by of the adenovirus genome, and wherein the only adenoviral sequences present in the vector are said 5'-inverted terminal repeat, said 3'-inverted terminal repeat, said packaging signal, and said nonexpressed segment of the E4 region.
The "E4 non-coding segment" refers to the nonexpressed segment of the E4 region co-localized with the E4 promoter. The segment is adjacent to the 3' TTR and, in human adenovirus serotype 5, is about 300 by in length.
Another aspect of the present invention describes a helper-dependent adenovirus vector having a GC content between about 50°70 and about 60%. The vector comprises in a 5' to 3' direction: (a) a 5' ITR; (b) a packaging signal cassette directly joined to the 3' of the 5' TTR; (c) a first stuffer DNA at least about 1 kb; (d) at least one heterologous expression cassette; (e) a second stuffer DNA at least about 1 kb; (f) an optionally present non-coding E4 segment; and (g) a 3' ITR. Element (f) if present is directly joined to element (g). The virus does not encode one or more adenovirus proteins needed for adenovirus generation and is about 28 kb to about 36 kb. Preferably, the virus does not encode any adenovirus proteins.
"Directly joined" indicates the absence of significant intervening sequences (e.g., less than about 150 bp) that interfere with the function of "joined"
groups.
"Stuffer sequences" refer to nucleic acid regions that do not express protein when present in a helper-dependent adenoviral vector. Preferably, such regions are mammalian non-gene regions or introns.
Another aspect of the present invention describes a cell line infected with a helper virus, a helper-dependent viral vector, or both the virus and the vector.
Preferably, the cell line expresses El proteins and a recombinase, and is infected with a helper virus that (1) contains an excisable low homology signal packaging cassette, and (2) does not express El proteins needed for viral generation.
Another aspect of this invention is a method of generating helper-dependent adenoviral gene vectors in a cell line expressing E1 proteins and Cre recombinase comprising:
a) infecting the cell line with a helper-dependent vector comprising: a 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present;
b) infecting the cell line with a helper virus comprising: an adenovirus genome having an E1 region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.
Another aspect of the present invention describes a method of generating helper-dependent adenoviral vectors. The method involves producing a cell comprising: (i) traps functions needed for adenovirus generation and (ii) a helper-dependent adenoviral vector comprising the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, wherein the helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.
The cell is used to generate the helper-dependent vector.
Reference to a cell comprising the traps functions needed for adenovirus generation indicates such functions are present in the cell, but need not be part of the cellular genome. The traps functions can be provided, for example, by a helper virus, the cellular genome, or a combination of both. Another example of a source of traps functions is a plasmid.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples.
The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention.
Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the shuffling of fragments of a stuffer to prevent genomic integration of the expression units.
WO 00/46360 PCT/iJS00/02405 FIGURE 2 is a gel showing restriction digests of helper-dependent adenoviral vector DNA after competitive rescues of: a) stk120gfp-E4-promoter and stk120gfp; b) stk120gfp-E4 and stk120gfp; and c) stk120gfp-E4-promoter and stk 120gfp-E4.
FIGURE 3 is a gel showing restriction digests of helper-dependent adenoviral vectors grown with the helper AdLCBBHG and a helper of this invention.
FIGURE 4 shows the structure of different helper-dependent adenoviral vectors.
FIGURE 5 shows autoradiographs of labeled restriction fragments from helper-dependent adenoviral vectors. The figure illustrates the results of competition experiments involving groups of helper-dependent adenoviral vectors from transfection to passage 6. "A" is a competition of backbones containing the shuffled fragments of cosmid HUMDXS455A (C4). "B" is a competition of backbones containing part of the HPRT (hypoxanthine guanine phosphoribosyltransferase) genomic gene. "C" is a competition between 2 backbones one containing part of the HPRT genomic gene the other containing the shuffled fragments of HUIVIDXS455A (C4).
FIGURE 6 is a graph showing the correlation between GC content and helper virus contamination.
FIGURE 7 is a graph showing the balance between helper and helper-dependent adenoviral vectors at different time points after infection. The figure demonstrates the replication potential of different helper-dependent adenoviral vectors with the mouse Erytropoetin gene relative to the helper virus.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is particularly useful for obtaining and isolating helper-dependent adenoviral vectors produced using a helper virus. Co-cultivation of a helper-dependent adenoviral vector and a helper virus produces two different particles competing for packaging and results in a mixed viral population. The production of the helper virus in the mixed viral population reduces the amount of helper-dependent adenoviral vector produced, provides a viral contaminant that interferes with applications of the helper-dependent adenoviral vector, and produces a source of nucleic acid that can be recombined with helper-dependent adenoviral vectors As described herein, production and isolation of helper-dependent adenoviral vectors can be facilitated in different ways. One way is to employ helper-dependent adenoviral vectors containing elements favoring viral generation of the helper-dependent adenoviral vector over a co-cultured helper virus. Another way is to employ helper viruses containing elements that can be employed to reduce the ability of the helper virus to grow, and /or recombine with the helper-dependent adenoviral vector.
The term "vector" employed herein is used in its broadest sense, and is meant to encompass a linear virus which can carry a heterologous gene, and or a plasmid which can contain one or more regions of a virus genome. The term encompasses a helper-dependent adenoviral vector and a helper virus.
Adenovirus components present in a helper or a helper-dependent vector may in general be provided from any adenovirus. Preferably, a human adenovirus serotype is used; more preferably, human adenovirus serotype 1-47;
and more preferably, a group C serotype (e.g., serotypes 1, 2, 5, and 6).
Preferred group C
serotypes are human adenovirus serotype 2 or 5, and more preferably serotype 5.
A helper-dependent adenoviral vector preferably contains a heterologous expression cassette. Such a cassette is made up of a heterologous gene functional coupled to a promoter and regulator elements needed for gene expression, and is particularly useful for introducing nucleic acid into a cell. The introduced nucleic acid preferably encodes for a gene or is able to regulate gene expression.
Nucleic acid able to regulate gene expression include ribozymes and antisense nucleic acids.
The ability to introduce nucleic acid into a cell using the present invention has different applications such as in gene therapy, in antibody production (e.g., a vaccine) and research to examine the regulation of a gene in vivo or in vitro.
Different genes can be introduced into a cell such as Factor VIII, Factor VIX, CFTR, OTC, LDL, VEGF, FGF, EPO, HSV-TK, IL-2, IL-12, p53, HLA-B7, a-interferon, and cytosine deaminase.
A promoter is a DNA sequence directing the synthesis of RNA through an RNA polymerase. Suitable promoters depend upon the gene and intended use, and may include promoters such as EFIa, CMV, chicken a-actin (Arnold, et al., Nucleic Acids Res 1988 Mar 25;16(6):2411-29), and muscle creatine kinase (Johnson, et al., Mol Cell Biol 1989 Aug;9(8):3393-9 ).
-g-WO 00/46360 1'CT/US00/02405 Generally, the regulatory elements that are present include a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal. Regulatory systems are available to control gene expression, such as GENE-SWITCHT~'' (Wang, et al., Gene Ther. 1997 May;4(5):432-41, U.S. Patent No. 5,874,534 and International Publication WO 93/23431, each of which are hereby incorporated by reference herein) and~those involving the tetracycline operator (U.S. Patent Nos. 5,464,758 and 5,650,298, both of which are hereby incorporated by reference herein).
A helper-dependent adenoviral vector is preferably from about 28 kb to about 36 kb to provide for efficient packaging. An adenovirus can accommodate up to about 105°10 of the wild-type genome and has a lower packaging limit of about 75% of the wild-type genome. (See, Parks, et al., J. Virology 71(4):3293-3298, 1997, hereby incorporated by reference herein.) In a preferred embodiment, there are no bacterial plasmid-based sequences (such as an origin of replication or bacterial marker genes) present in the helper-dependent adenoviral vector.
Construction of helper viruses and helper-dependent viruses can be achieved based on the guidance provided herein using techniques well known in the art, such as those described by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A
laboratory Manual, 2°d Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein. Additional descriptions of useful techniques are provided, for example, in the Example section provided below and references such as Mitani, et al., Proc. Natl. Acad. Sci. USA 92:3854-3858, 1995; Fisher, et al., Virology 217:11-22, 1996; Kochanek, et al., Proc. Natl. Acad. Sci. USA 93:
5736, 1996; Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996;
Parks, et al., J. Virology 71 (4):3293-3298, 1997; and Schiedner, et al., Nature Genetics 18:180-183, 1998; each of which are hereby incorporated by reference herein.
The helper-dependent adenoviral vector and the helper virus can be generated in a wide range of host cells able to be infected by adenovirus.
Examples of host cells include 293 cells, 911 cells and PERC.6 cells (WO 97/00326 hereby incorporated by reference herein). The employed cell can be constructed to provide for useful proteins such as viral proteins or a recombinase.
HELPER VIRUS
Helper virus elements useful for facilitating the production and isolation of helper-dependent viruses include a low homology excisable packaging signal cassette and a substituted E3 region. Preferred helper viruses contain both a low homology excisable packaging signal and a substituted E3 gene.
Low Homolo~y Excisable Packaging-Signal Cassette The low homology excisable packaging signal cassette contains a modified packaging sequence which fulfills the role of an adenovirus packaging signal, has a low homology to the Adenovirus packaging signal, and is flanked by a 3' and 5' recombinase recognition sequence. Preferably, the modified packaging signal is less efficient than a wild type packaging signal. Advantages of such low homology excisable packaging signals include: (1) less recombination between the helper virus and helper-dependent adenoviral vector; and (2) when a less efficient packaging signal is used, less packaging of those helper viruses which escaped excision of the packaging signal.
Recombinase recognition sequences recombine when acted on by a recombinase that recognizes the sequences, resulting in the excision and circularisation of intervening nucleic acid. The recognition sequences should be sufficiently far apart, at least about 180 bp, to allow for the proper positioning of nucleic acid segments being recombined. Preferably, the recombinase recognition sequence is loxP or frt.
The loxP recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Cre recombinase. Cells, such as 293 cre cells, stably expressing Cre recombinase can efficiently remove nucleic acid located between loxP
recognition sites. (See, Parks, et al., Proc. Natl. Acad. Sci. 93:13565-13570, 1996 and Parks, et al., J. Virology 71 (4):3293-3298, 1997, both hereby incorporated by reference herein).
The frt recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Flp recombinase. Cells stably expressing Flp recombinase can efficiently remove nucleic acid located between frt recognition sites.
Flp-mediated gene modifications are described in U.S. Patent No. 5,564,182, hereby incorporated by reference herein.
Homology between a helper and the helper-dependent adenoviral vector encourages recombination events between the two, resulting in unwanted changes in the structure of the helper-dependent adenoviral vector or the helper virus, and leading to an increased contamination by helper virus. For example, if the first of a loxP site flanking the packaging signal is removed by homologous recombination within the packaging signal, the resulting helper virus escapes selection in 293cre cells because the Cre recombinase is no longer able to excise the packaging signal.
Sequences for different low homology excisable packaging signal cassettes can readily be designed taking into account recombinase recognition sequences and adenovirus wild-type packaging signal sequences using the guidance provided herein. The provided guidance focuses on the adenovirus serotype 5 packaging signal to illustrate the production of a modified packaging signal.
Such guidance is applicable to producing other modified packaging signals taking into account other adenovirus serotypes.
The wild-type packaging signal of adenovirus serotype 5 is formed by at least seven functional units called A repeats, which are located between nt 230 and nt 380 of the genome. The A elements have the consensus sequence ATTTGNBGC
(Schmid et al, 1997 J. Virol. 71:3375-3384, which is hereby incorporated by reference). The modified packaging signal of this invention preferably comprises less packaging elements than the wild-type (which has seven elements), preferably from about two to six elements, and more preferably from three to five elements. In a preferred embodiment, the modified packaging signal contains only four out of the seven original packaging elements (elements AI to AIV). The four elements are preferably in a modified form with two strong elements (AI and AII) present.
The position of the elements relative to each other may be changed, but in preferred embodiments, it is maintained.
In order to reduce contiguous sequence homology, the eight ambiguous nucleotides of the consensus sequence (ATTTGNBGC; SEQ. ID. NO. 1) within each A element are preferably replaced by sequences taken from a different A
element.
For example, the eight nucleotides within A1 were replaced by those from AV;
and the eight nucleotides within All were replaced by those from AVI. In addition, a new element was created between All and AIII starting 21 by after AII, by changing the existing nucleotides to the consensus sequence. Two more nucleotides were exchanged within AIV: ATTTTGTGTT (SEQ. ID. NO. 2) was changed to ATTTTGTTGT (SEQ. ID. NO. 3). One embodiment of a synthetic packaging signal is given in SEQ. ID. NO. 4.
Desired nucleic acid sequences can be produced using different techniques including those involving the creation of nucleic acid mutations and nucleic acid synthesis techniques. Examples of such techniques are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A laboratory Manual, 2°'~
Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein.
F~ Tnec~rtinn Homologous recombination between viral sequences (E1) present in the cell lines used for propagation of either helper virus (lacking El) or helper-dependent adenoviral vector, and the helper virus itself, has resulted in the generation of wild-type viruses. Homologous recombination results in the insertion of the region into an El-deleted virus genome. To avoid this problem, in preferred embodiments of this inventions, the helper virus also contains an insertion element.
Preferably, the insertion element does not contain a promoter or encode for a protein when present in the helper virus. More preferably, the insertion element is non-coding repeat-free human DNA devoid of splice signals.
Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996, describe the use of an E3 region containing an insert encoding for the ampicillin gene, the bacterial origin of replication, a CMV promoter and the luciferase gene (AdLC8BHG101uc). The insert protected against the production of viable viruses having a wild type E1 produced by homologous recombination. However, the insert impairs virus generation, most likely by interference with fiber gene expression. The resulting level of viral generation is disfavored for the production of viral stock.
The use of non-coding sequences as an E3 insertion element, preferably human non-gene sequences, provides for more efficient viral generation than that observed with E3 insertional elements encoding for bacterial protein described by Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996 (see Table 1, infra.) The use of intron sequences are preferred because such sequences do not contain hidden splice signals that could interfere with correct splicing of the fiber message.
The size of the insertion element (preferably between about 2800 and 3500 bp, more preferably about 2900 bp) is chosen to avoid the generation of potentially harmful wild-type viruses while maintaining a virus size close to wild-type size. Starting with a helper virus having a deleted E1, if homologous recombination occurs resulting in insertion of the El region, the recombined adenoviral vector which also contains the E3 insertion element would have a genome size of approximately 38350 by (108.1% of wild-type size). Because this size exceeds the packaging limit of Adenovirus 5, no viable virus would be produced.
The insertion element may be from virtually any source. In a preferred embodiment, a 2900 by insert was taken from human chromosome l 1q13, starting 13340 by upstream of STS marker 11S1337 (available from Genethon) and inserted into the Xba I site at position (nt) 28593 (referring to Ad5 wild-type).
The insertion element and its insertion point in E3 should be chosen to avoid any reduction in synthesis of the fiber protein. Such a reduction is likely to occur if the E3 region is modified because synthesis of fiber protein is dependent on correct splicing of RNA from signals located in this region. We demonstrate that viruses containing the insert grow to titers similar to those obtained with viruses containing an unchanged E3 region.
HELPER-DEPENDENT ADENOVIRAL VECTOR
Helper-dependent adenoviral vector elements useful for facilitating the production and stability of helper-dependent adenoviral vectors include an E4 non-coding segment that confers a selective advantage to a virus and stuffer regions) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. Preferably, the helper-dependent adenoviral vector contains an E4 non-coding segment conferring a selective advantage to the vector; and stuffer regions) providing the vector with a GC content of about 50% to about 60% and an overall size of about 28 kb to about 36 kb. In a preferred embodiment, there are no bacterial plasmid-based sequences (such as an origin of replication or bacterial marker genes) present in the helper-dependent adenoviral vector.
Stuffer DNA
Stuffer sequences are used to provide a helper-dependent adenoviral vector with an overall size of about 28 kb to about 36 kb. A helper-dependent adenoviral vector containing only a heterologous expression cassette, adenoviral ITR's and a packaging signal may be quite small, in general about 5-10 kb, depending on the size of the heterologous expression cassette. Because such a small virus does not package efficiently stuffer sequences are added to provide a final size of about 28 kb to about 36 kb. Preferably, the stuffer provides the vector with a GC
content of about 50% to about 60%.
Stuffer sequences used in gene therapy applications should not contain active genes or be recognized as foreign in a human cell. A preferred stuffer sequence replicates as well as the adenovirus itself, is not recognized by the host as foreign DNA and does not lead to chromosomal integration of the transgene.
Genomic DNA of mammalian origin, or even more preferably of human origin, are preferred stuffer sequences for gene therapy applications to avoid eliciting an immune response. However, stuffer based on human genomic DNA is identical to chromosomal sequences in a human target cell and could allow for insertion of the heterologous expression cassette into the host genome by homologous recombination. This phenomenon could have a potentially dangerous effect and should be prevented.
To prevent integration of the heterologous expression cassette into the host genome, the contiguous stuffer sequence can be interrupted, the individual fragments reversed and the expression units inserted at the specific breakpoints. (See for example, Figure l.) Using this strategy, the transgene is not flanked by regions which would support homologous recombination. Additionally, potentially unstable genetic elements such as retrovirus LTR as well as genomic repeats (e.g., MIR, and ALU) should avoided or removed to prevent rearrangements during amplification.
Another consideration is the GC content of the virus. The GC content that stuffer sequences provide to a helper-dependent adenoviral vector has a major impact on yield and purity of the virus. Competition experiments between different viruses as well as individual analysis revealed a correlation between the GC
content of a virus and growth properties. The GC content is preferably between about 50%
and about 60% and even more preferably between 52% and 57% which is close to the GC of wild-type adenovirus. Without being limited to any particular theory, advantageous growth properties are believed to be based on more efficient generation of a virus with a higher GC content.
Preferably, stuffer sequences are provided such that the heterologous gene cassettes) that are present are at least about 1.0 kb from the helper-dependent viral vector 3' end and 5' end, more preferably at least about 2.0 kb, and more preferably at least about 4.0 kb. Additionally, high AT rich segments such as those provided in the hprt fragment (59% AT) may be unfavorable even with a viral vector having an overall high GC content. A "high" AT is above about 55% AT.
E4 Non-coding Segment The E4 region of adenovirus type 5 occupies about 3000 by at the right end of the genome. Transcription from the E4 promoter is directed from the right end toward the center. Seven polypeptides are produced from open reading frames (ORFs) located within the region with the first ORF starting at nucleotide (nt) 406 from the right end of the virus.
It was observed that the E4 region is sometimes transferred from a helper to a helper-dependent adenoviral vector as a result of homologous recombination. This recombined helper-dependent adenoviral vector generally is favored in co-cultivation with the non-recombined helper-dependent adenoviral vector and dominates in vector preparations. Despite its strength, this recombined helper-dependent adenoviral vector cannot be used as a helper-dependent vector for gene therapy because it contains functional E4 genes. Expression of those E4 genes in a host cause an immune rejection of vector infected cells by the host.
In accordance with this invention, it has been determined that a noncoding segment of the E4 region confers a selective advantage when a virus is co-cultivated with a virus lacking the same element. The entire E4 region is not needed.
From adenovirus serotype 5, the part of E4 between the 3' ITR (starting 102 nt from the right end) and nt 405 which does not contain coding sequence is the segment that should be preferably included into the helper-dependent vector and is a non-coding E4 segment. E4 sequences located left of this element have no additional impact on virus strength. This is of importance because all genes (open reading frames, orfs) are located in the left part of the E4 region. Thus, another aspect of this invention is a cis-acting sequence located next to the 3' ITR (right virus end) which is preferably incorporated into the helper-dependent adenoviral vector because it supports amplification of the helper-dependent adenoviral vector.
Competition experiments were designed to determine the location of the beneficial element. Helper-dependent adenoviral vectors based on the vector stk120 (which contains only a 5' ITR, a 3' ITR and packaging signal as its viral-based nucleotides, as described in Morsy et al., 1998, Proc. Natl. Acad. Sci. USA
95:7866-7871, which is hereby incorporated by reference) were constructed containing either the 3' ITR only (stk120gfp), the 3' TTR and noncoding sequence of the E4 region until nt 400 from the right end (stk120gfp-E4promoter ) or the 3' ITR and the complete E4 region to nt 3115 from the right end (stk120gfp-E4). Pairs of these vectors were cotransfected at a 1:1 ratio to originate rescues of two competing helper-dependent adenoviral vectors.
After passage seven, viruses from at least three independent rescue procedures were purified, and DNA was extracted and analyzed by radioactively labeled restriction digests. The two competing helper-dependent adenoviral vectors have an almost identical restriction pattern. The only difference is in the size of one fragment, called the discriminating fragment. The discriminating fragment was found 5-10 fold stronger for stk120gfp-E4-promoter than for stk120gfp, and at least 4 fold stronger for stk120gfp-E4 than for stk120gfp (see Figures 2a and 2b). However, the discriminating fragments are of equal strength for stk120gfp-E4-promoter and stk120gfp-E4 in each rescue (see Figure 2c). Therefore an E4 element supporting helper-dependent adenoviral vector growth is present in stk120gfp-E4 promoter and stk120gfp-E4 and must be entirely located in the right part of the E4 region (between nucleotide -400 from the right end and the 3' ITR).
While not wishing to be bound by theory, it appears that there are two possible explanations for the selective advantage to the E4 segment-containing adenoviral vector: the E4 element allows for more effective replication of the virus or it confers improved packaging and stability. Our results indicate that the E4 segment does not effect replication. If infection is started with equal amounts of helper and helper-dependent adenoviral vectors, the ratio between helper and helper-dependent genomes inside the cell at the end of the production cycle (48 hours) is identical.
However, 3-fold more helper-dependent adenoviral vectors were packaged for the genome containing the E4 segment compared to contaminating helper genomes.
Thus, it appears that the E4 segment supports effective packaging.
EXAMPLES
Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
General Materials And Methods Construction Of Plasmids Cloning techniques described in J Sambrook, E.F. Fritsch and T.
Maniatis, 1989 Molecular Cloni~zg, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference, were used.
To generate plasmids containing the entire Ad5 genome, homologous recombination in E. coli based on the recF pathway was used. Competent cells of E.
coli strain BJ 5183 were transformed with a plasmid vector containing the adenovirus genome and a fragment from the shuttle vector. This fragment contains the sequence to be inserted flanked on both sides by sequences homologous to the vector. In order to select against re-transformation of the vector without modification, it was linearized close to the insertion point. DNA isolated from individual colonies of BJ5183 was retransformed into the strain XL2 (Stratagene) and high yield plasmid preparations were obtained. Correct clones were determined by restriction analysis and sequencing.
Virus Rescue 10~.g helper plasmid was digested with PacI and transfected by calcium phosphate coprecipitation into a 6 cm dish of subconfluent 293 cells.
Directly after transfection cells were overlaid with aMEM/ 10% FCS/ 0.8 %
Seaplaque agarose. 20-100 plaques appeared after 7-9 days. Plaques were taken days after transfection and used to reinfect cells. After two intermediate passages the cell lysate was used to infect NUNC-cell factories (NUNC). Cells were harvested 48 hours after infection and virus was released by three freeze/thaw steps steps (-70°C
/37°C). After treatment of the cleared lysate with BENZONASE nuclease, viruses were purified by ultracentrifugation on a CsCI gradient. After one step gradient (q=
1.5 / 1.35 1.25 g/cm3) the viruses were loaded on a continuous gradient formed from a CsCI solution of q= 1.35 g/cm3. Viruses were dialyzed against IOmM Tris pH
8.0, IOmM MgCl2 and 10% glycerol and stored in aliquots at -70°C. Helper-dependent adenoviral vectors were purified using substantially the same procedure. For analytical purposes virus obtained from two 15 cm dishes was purified. Virus DNA
was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using aP33dATP or dCTP and Klenow polymerise and separated in 0.7%
agarose gels. The gel was dried and exposed to film.
Determination Of Particle Titer lOp,l of virus suspension was added to 90,1 PBS/ 0.1 % SDS and incubated at 50°C for 20 minutes. OD260 is determined spectrophotometrically and the concentration of virus particles was calculated based on the equation 1 OD260 =
1.1x1012 particles.
Extraction Of Viral DNA and Restriction Digest 100.1 of virus suspension were added to 100,1 lysis solution (IOmM
Tris-HC1 pH 7.5, lOmM EDTA, 0.5% SDS, 0.05% pronase) under constant vortex.
After an incubation at 37°C for at least 2 hours followed by addition of 100,1 TE the lysate was extracted lx with Tris-HCl saturated phenol. Virus DNA was precipitated with two volumes of ethanol, washed extensively with 70% ethanol and resuspended in TE. Virus DNA was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using a,P33dATP or dCTP and Klenow polymerise and separated in 0.7% agarose gels. The gel was dried and exposed to film.
Extraction of Cellular DNA From Infected Cells 10~ cells are lysed inlOmM Tris-HC1 pH 7.5, IOmM EDTA, 0.5% SDS
and treated with 100~.g/ml Proteinase K overnight at 58°C. The lysate was extracted with Tris saturated phenol, phenol/chloroform/ isoamylalcohol (24:24:1) and chloroform, precipitated with two volumes of ethanol, washed extensively with 70%
ethanol and resuspended in TE.
End Point Dilution Assay 96 well plates were seeded with 1x104 293 cells in 100,1 per well 24 hours before the assay. Each virus stock was diluted 103 to 108- fold.
Dilutions were used to infect 24 wells each (50,1/ well). Positive wells were scored by cytopatic effect after 14 days and the virus titer was calculated based on two dilutions with intermediate numbers of positive wells taking the dilution factor into account.
Quantitative PCR
Real time quantitative PCR (ABI PRISM 7700) was used to determine the relative amounts of helper and helper-dependent adenoviral vector. Two specific target sequences were selected present either in all helper viruses (Ad5 sequences from 11358-11456) or in all helper-dependent adenoviral vectors based on stk120 (bp 12037-12176) (Morsy et al., 1998, Proc. Natl. Acad. Sci. USA 95:7866-7871). A
plasmid containing both target sequences was constructed and used as one standard for both amplifications. Within each target sequence, a set of forward primer, reverse primer and probe (which is located between the primers and contains a fluorogenic reporter and a quencher) have been selected. Separation of quencher and reporter generated a fluorogenic signal during the logarithmic phase of PCR
amplification which was plotted versus cycle number. The plot was used to calculate template concentration based on the software of the 7700 system (ABI).
Construction Of Helper Viruses Helper viruses for the helper-dependent system were made based on AdS. In order to obtain specific helper functions, the plasmid pAdEl-E3+
(containing the entire Ad5 genome with a complete deletion of the E1 region) was modified in two regions: i) between the left ITR and the promoter of protein IX; and ii) in the E3 region.
To introduce changes at the left end of the genome a shuttle vector "ploxOpack" was constructed. It is based on pUC (Boehringer Mannheim) and contains the following elements:
A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt 1;
B) a loxP site starting directly after nt 195 of AdS;
C) a synthetic packaging signal contained in the following sequence:
5'- GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA
TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC
GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA
ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG-3' (SEQ.ID.N0:4).
D) a second lox P site;
E) a linker containing cloning sites; and F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
For comparison, a second shuttle vector, "ploxpack", containing the wild type packaging signal instead of the synthetic one was constructed. It contained:
A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt 1;
B) a loxP site starting directly after nt 195 of AdS;
C) Elements AI-AIV of the wild-type packaging signal contained in the following sequence:
5'-GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG
GCGGATGTTGTAGT AAATTT GGGCGTAACC GAGTAAGATT
TGGCCATTTT CGCGGGAAAACTGAA TAAGA GGAAGTGAAA
TCTGAATAAT TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG-3' (SEQ.
>D. NO. 5) D) a second lox P site;
E) a linker containing cloning sites; and F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
The second shuttle vector was generated by cloning the 4.9kb XhoI
fragment from Ad5 wild-type (AdSwt} DNA (nt 24797-29791) into pUC. A 2900 by fragment of human DNA located on chromosome) 1q13 starting 13340 by upstream of STS marker 11S1337 (Genethon) was obtained by PCR and inserted into the XbaI
site at nt 28593 (AdSwt) in either orientation. This fragment contains part of inton 2 of the human LRPS gene.
A strategy based on homologous recombination between plasmids in E. coli (Chattier, et al., 1996 J. Virol 70: 4805-4810, which is hereby incorporated by reference) was used to introduce those sequence elements into plasmid pAd5E1-E3+
containing a complete Ad5 genome with a deletion of El. The procedure allowed for insertion of those elements at exactly the positions described for the shuttle vectors.
The flanking Ad5 derived sequences were completely maintained. The following plasmids for new helper viruses were constructed:
pAdlspLI1 = Helper 14 -- has synthetic packaging signal, intron of LRPS in E3;
pAdlpLI1 = Helper 1 -- has wild type packaging signal, intron of LRPS in E3;
pAdlpE3 = Helper 11- -has wild type packaging signal, wild type E3.
Plasmids containing the Ad 5 genome with the described modifications were digested with PacI to release the virus genome from the plasmid and transfected into 293 cells by calcium phosphate coprecipitation. Resulting virus plaques were used to reinfect cells. Amplified virus stocks were characterized by restriction and sequence analysis.
1. Growth of helper viruses in 293 and 293cre cells Different helper viruses as well as a El deficient adenovirus (d170-3) (Bett et al. Proc. Natl. Acad. Sci. USA 91:8902-8906 which is hereby incorporated by reference) taken as control, were grown in 10 layer NUNC cell factories, purified in CsCI Gradients and dialyzed. The virus concentration (particle concentration) was determined by spectro-photometric analysis (OD260). 1.5 x 106 cells (293 or cre) were infected in a 6 cm plate with 100 particles per cell. CPE was apparent in all plates after 48 hours. Cells and medium were harvested, frozen and thawed 3x and the titer was determined in an end point dilution assay. The number of infectious viruses produced per cell was calculated and the results are shown in Table 1.
Table 1 d170-3 AdLCBBHGlOIuc AdIpLII AdlpLI1 Ad without Helper Helper 14 lox 1 Wt. 'IJ Synth. 'll 293cells 1200' 27 840 610 293cre4 cells1400 1.2 1.3 0.8 *infectious units per cell The amplification rate for all new helper viruses exceeds that of AdLC8BHG101uc (Parks et al., 1996, Proc. Natl. Acad. Sci. USA 93:13565-13570, which is hereby incorporated by reference) by at least 20 fold. The growth of the helper viruses in contrast to d170-3 is well controlled in 293cre4 cells.
These cells release only 1-2 infectious particles/cell. The rate of suppression is therefore dramatically increased for the new helper viruses. Viruses without or with the insert in E3 in either orientation do not differ much in virus yields; thus, the chosen E3 insert did not have a major impact on virus growth.
2. Amplification of helper-dependent adenoviral vectors over a single passage A characterized stock (known particle titer and helper contamination) of a helper-dependent adenoviral vector containing the human OTC gene was used to infect 293cre cells. Cells were infected with 700 particles of the helper-dependent adenoviral vector and 100 particles of one of the helper viruses per cell.
After 48 hours cells were collected, virus was released through 3 freeze/thaw steps and purified by Cs banding. Particle titers were determined by spectrophotometric analysis (OD260). Virus DNA was extracted and analyzed by restriction digests (Figure 3).
Overall virus yield was slightly increased for the new helper virus over helper AdLC8BHG101uc. However, the ratio between DNA-containing particles and empty particles is increased for the new helper. Since helper DNA is undetectable in the gel, helper virus does not contribute significantly to the overall output.
To determine helper contamination more accurately, quantitative PCR
based on TaqmanT"'' technology was used. Contamination was similar between the helper AdLCBBHGIOIuc and the helper LPLI1 containing the original packaging signal, whereas the amplification supported by helper LSPLI1 containing the modified packaging signal resulted in a lower contamination (0.29%) (Table 2). This is expected because yields of virus grown in 293 cells are lower than those of an identical virus containing the normal packaging signal. Therefore helper virus which would escape cre-selection should still have a selective disadvantage.
Table 2 HD Virus Helper Contamination Producti vit %cont. St. Dev. %RSD
AdLCBBHGlOIuc 4800 0.52% 0.1% 22%
Helper 1 (Wt. 5300 0.42% 0.12% 28%
'Y) Helper 14 (Synth.'I')8100 0.29% 0.06% 21%
3. Construction of modified backbones Constructions are based on plasmid stk120 (Morsy et al., 1998, supra) containing a CMV promoter driven gfp (green florescent protein) gene within the stuffer sequences at a unique SwaI site (stk120gfp). In order to incorporate the additional elements into the helper-dependent adenoviral vector, the 3'ITR
(130 bp) was extended to 400 by from the right end of the Ad5 genome. This fragment contains the complete E4 promoter region. However, no adenovirus coding sequences are present. The resulting adenoviral vector is stk120gfp-E4-promoter.
In a second construct, a 1353 by fragment from the right end of stk120gfp containing part of the stuffer sequence and the 3' ITR was replaced by a 3115 by fragment of Ad5 from a MunI site to the end of the genome (stk120gfp-E4).
This fragment contains the complete functional E4 region.
4. Competition between helper-dependent adenoviral vectors Genomes for stk120gfp and stk120gfp-E4-promoter were released from the respective plasmids and cotransfected with a circular plasmid containing the entire helper genome into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus at moi 1.
The mixture of viruses was amplified up to passage 7 where the viruses were Cs-purified. Five independent experiments were carned out. Virus DNA was extracted and analyzed for presence of the two virus types. In all experiments a 5-10 fold excess of stk120gfp-E4-promoter was found (Figure 2a).
This indicates the presence of a cis-acting sequence located next to the 3' ITR (right virus end) which supports helper-dependent adenoviral vector growth.
An identical experiment was carried out to determine whether the complete element is located within 400 by from the right virus end or sequences further upstream contribute to this feature. stk120gfp-E4-promoter and stk120gfp-E4 were cotransfected with the circular plasmid containing the entire helper genome (Helper 14) into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus 36 hours after transfection at moi 1. After passage 7, viruses from three independent rescue procedures were purified, DNA was extracted, digested with BsrGl and labeled. The discriminating fragments have equal strength for each rescue (Figure 2c). Therefore, the cis-acting sequence supporting helper-dependent adenoviral vector growth is located between nucleotide -400 from the right end and the 3' ITR.
Example 3 Construction Of Helper-Dependent Adenoviral Backbone Containin~lAlternative Stuffer Sequences The construction of the new helper-dependent adenoviral vectors (group A) began with the ligation of a 3498 by from fragment from pSTK120 (which comprised of the bacterial sequences origin of replication and ampicillin resistance gene, the right and left TTRs and the packaging signal) ligated with a multiple cloning site linker (SEQ. ID. NOs. 6 and 7) containing HindI>I sticky ends and cloning sites EagI, AscI, BgIII, NotI, Xbal, Mlul, EcoRl and Kasl. This clone is named "pITRF"
DNA from the region encompassed within HUMDXS455A (Genebank Accession # L31948) used as stuffer DNA was amplified from human genomic DNA
in four segments of about 4.5kb each using the following pairs of primers:
SEQ. 1D NOs. 8 and (PCR
9 1 ) SEQ. ID NOs. 10 (PCR2) and 11 SEQ. ll~ NOs. 12 and (PCR3) SEQ. >D NOs. 14 and (PCR4) PCR1 product was Klenow filled in and then digested with Ascl. It was ligated directly into the Ascl/EcoRV of pITRF to produce "Construct 1".
PCR2 product was digested with Notl/Ascl and cloned into the Notl/Ascl site of Construct 1 to produce "Construct 2". PCR3 product and Construct 2 were digested with Mlul/Notl (double digest) and ligated to produce "Construct 3".
The PCR4 product was first cloned into a TA cloning vector.
To avoid inefficient direct cloning for very large plasmids homologous recombination in E. coli BJ 5183 was used to insert PCR4 and additional genomic fragments. The technique requires a small shuttle vector containing the region which overlaps with the insertion point in the large plasmid. This shuttle vector was constructed as follows: 2 kb of the junction over PCR3 and vector sequence was amplified using Construct 3 DNA. The PCR fragment was Klenow filled in and then digested with Sapl enzyme and ligated into PABS helper-dependent-3.0, a small plasmid vector (KanR) cut with EcoRI followed by a fill in reaction using Klenow and a digestion with Sapl. Homologous recombination between Construct 3 and the shuttle resulted in Construct 4 (C4).
The helper-dependent adenoviral vector part of C4 is 20kb in length and has 3 unique restriction enzyme sites to incorporate 3 different transgenes (Ascl, Notl and Swal).
As a next step, a CMV promoter driven gfp gene was inserted into the NotI site of C4 by homologous recombination. For this purpose, a shuttle vector was built containing 2 kb of C4 overlapping the NotI site (pNot). The gfp expression unit was inserted by blunt end cloning into NotI and a fragment of this vector was used for homologous recombination with C4. Because a single NotI site is also present in the gfp expression unit, the gene can later be replaced by other genes using this restriction site. The gfp gene is located at a junction between 2 inverted genomic fragments which prevents insertion into the host genome by homologous recombination which would be likely, if the gene was inserted within a contiguous sequence.
To incorporate additional stuffer sequences and extend the 3' TTR to include the E4 promoter, a second shuttle based on pABSHD-3 was constructed containing 2 kb of C4 including the 3' ITR and unique KasI, SaII, BgIII and HindIII
sites for insertion. The E4 promoter was directly cloned as a Hind III/ SaII
fragment into this shuttle vector.
Stuffer DNA fragments AFO, HSU, ERl, and ER2 (Figure 4), were amplified from human genomic DNA by PCR Expand Kit (Boehringer) and cloned into the Topo-blunt vector (Invitrogene). AFO and HSU were inserted individually as SaII fragments, the ER1 and ER2 fragments were inserted sequentially after KasI/
BgIII and BgIII/ Sal digests. Recombination in E. coli BJ 5183 lead to the final backbones C4HSU (SEQ. >D. NO. 16), C4AF0 (SEQ. >D. NO. 17), and C4ER
(Figure 4).
Construction of the new helper-dependent backbones (group B) was started by replacement of a 8181bp fragment from stk120 by the gfp expression unit.
For this purpose a shuttle vector was built based on pUC 18 containing lkb 5' to the unique SwaI site and lkb 3' to the unique EagI site in stk 120. The gfp expression unit was inserted into the Bam HI site of this shuttle as a BgIII fragment.
The replacement was carried out by homologous recombination in E. coli BJ 5183 between STK 120 linearized with EagI and a fragment of the shuttle vector.
This step removes a part of the hprt region from the vector which was most frequently lost during adenoviral vector propagation. This vector "STKSEgfp" is 19.4 kb in length and misses about lOkb of genomic sequence required for propagation as a helper-dependent adenoviral vector.
Fragments AFO and HSU were cloned after SaII digestion into the Sal I site of shuttle vector pSHI-3ITR which contains part of the HUMDXS455A -fragment in STK120 and the 3' end of Ad5 starting from nt -400 containing the promoter and the 3' ITR. The fragments ER1 and ER2 were inserted step by step into the same vector as SaII /BamHl and BamHl/BgIII fragments. The 3 resulting different shuttle vectors were recombined with STKSEgfp linearized with PacI
to generate HXAFO, HXHSU and HXER (Figure 4).
Example 4 Generation Of Helper-Dependent Adenoviral Vectors Cells from subconfluent plates were washed with PBS, trypsinized and seeded to 6 well plates (Costar) at 3 x 105 cells per well in 1.5m1 medium.
Genomes for helper-dependent adenoviral vector were released from the respective plasmids by Pme I digestion and 2~.g of DNA were cotransfected with a 2~.g circular plasmid containing the entire helper genome per well. A 24 hour transfection was followed by infection with helper virus at 1 moi. Forty-eight hours later cells were lysed by 3 freeze/thaw cycles and the lysate was used to infect 106 293cre cells together with Helper virus at MOI 5. Cells were lysed 48 hours later.
The same procedure was repeated with increasing the number of cells until passage 6 at which two 15 cm plates are infected. At this stage helper-dependent adenoviral vector is isolated by CsCI banding and analyzed by restriction digestion.
Passage 6 is also used to infect a large scale preparation (1x10 layer Nunc cell factory).
Example 5 Competition Between Helper-Dependent Adenoviral Vectors Virus rescue in a competition experiment was performed using the procedures described above and, instead of DNA from a single vector plasmid, a equimolar mixture of 3 vectors was used. Three mixed rescues were carried out:
one with HXAFO, HXHSU and HXER; one with C4AF0, C4HSU and C4ER; and the third with HXHSU and C4HSU. No rearrangements were observed in either the individual or the mixed rescues.
As shown in Figure 5 there are only moderate differences between backbones within the first 2 groups with a slight dominance of the C4HSU
construct.
However, in a competition experiment between HXHSU and C4HSU the latter clearly dominated. Thus, the remaining hprt sequences in HXHSU have an adverse effect on adenoviral vector propagation.
Example 6 Analysis Of Large Scale Preparations Of Helper-Dependent Adenoviral Vectors STK120-EFIamEPO. C4AF0-EFIamEPO and C4HSU-EFIamEPO
To investigate the behavior of the new helper-dependent adenoviral vector in vivo, the gfp gene was replaced in STK120, C4AF0 and C4HSU by the in mice immunologically inert mEPO gene. Replacement was carried out using the shuttle pNot by homologous recombination. Viruses were rescued as described above. Large scale amplifications were carned out in Nunc cell factories and the virus was purified as described above.
Since the size of the helper-dependent adenoviral vector and the helper differ only slightly, the purification procedure does not remove helper virus from the preparation. Such a physical separation was not intended to allow for a replacement of gradient based by chromatography based purification in large scale. DNA was extracted from purified virus and the content of helper virus relative to the amount of helper-dependent adenoviral vector was determined by TaqmanTM quantitative PCR.
We observed a correlation between the helper content and the GC
content of the helper-dependent adenoviral vector (see Figure 6). The experiment with Construct C4HSU has the lowest helper content (about 0.18%) compared to a helper content of about 1 % for stk120.
Example 7 Analysis Of The Replication Potential Of Different Helper-Dependent Adenoviral Vectors Cells were infected with 100 part/cell of helper and 300 part/cell of the helper-dependent adenoviral vector. Three hours after infection 1/3 of the cells was harvested and washed with PBS. Another portion was harvested at 42 hours and a third fraction at 48 hours. Cellular DNA was extracted as described above and the relative amount of helper and helper-dependent adenoviral vector DNA was determined by TaqmanTM PCR (Figure 7).
Figure 7 shows that if the rescue is started with an excess of helper-dependent adenoviral vector, the ratio of helper-dependent/helper is maintained and even slightly increased for C4HSU, whereas it is strongly decreasing for STK120 and slightly decreasing for C4AF0. This shows that the nature of the backbone has a strong impact on the speed of replication. Whereas the Adenovirus genome coevolved with the adenovirus polymerase, the helper-dependent adenoviral vector sequence was artificially selected. Therefore a less efficient replication is expected.
However, the most important determinant seems to be the GC content of the helper-dependent adenoviral vector.
Example 8 Sequence Information The different sequences for the SEQ. ID. NOs. referred to in the application are as follows:
SEQ. ID. NO. 1 ATTTGNBGC
SEQ. m. NO. 2 ATTTTGTGTT
SEQ. >D. NO. 3 ATTTTGTTGT
SEQ. ID. NO. 4 GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA
TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC
GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA
ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG
SEQ. ll~. NO. 5 GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG GCGGATGTTGTAGT
AAATTT GGGCGTAACC GAGTAAGATT TGGCCATTTT
CGCGGGAAAACTGAA TAAGA GGAAGTGAAA TCTGAATAAT
TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG
SEQ. m. NO. 6 AGCTCGGCCGATTATTGGCGCGCCAGATCTGCGGCCGCTTCTAGAAACGC
GTGAATTCGGCGCCA
SEQ. ID. NO. 7 AGCTTGGCGCCGAATTCACGCGTTTCTAGAAGCGGCCGCAGATCTGGCGC
GCCAATAATCGGCCG
SEQ. ID. NO. 8 ATTGGCGCGCCTTCTTTCTGGGATGATTCAGCATCAACTC
SEQ. ID. NO. 9 GATCGTCGGCCGCTTGGGTCATAGACTTCTTTGAGAACCAG
SEQ. m. NO. 10 ATCAGTTAGCGGCCGCACAAGCTAAGATCACAAAGCTGTTT
SEQ. m. NO. 11 TATGGCGCGCCGCTGACACCCAGCCTGGGTGCCGGTG
SEQ. ID. NO. 12 TCGACGCGTAGCGCTGTGTGGCCTTGGCAGTTTCCATAG
SEQ. m. NO. 13 TCAGTAATGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTG
SEQ. m. NO. 14 TATTAAGGCGCCGGGCATGGGAGTGATCTCACCAACTCTGG
SEQ. ~. NO. 15 TCGACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGT
SEQ. ~. NO. 16 Helper Dependent vector C4HSU
The sequence is shown without transgenes.
AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG
AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG
ACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATG
TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA
CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT
GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA
GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC
TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT
TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC
GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC
CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT
ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC
TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG
GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG
CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG
AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA
GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA
AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC
CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT
CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC
ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA
TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC
CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA
GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA
AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC
TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA
ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC
TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT
ATTTTGATGTAATATTTGAAATAACAATAAAAATTGCCACCACTGAATTTA
TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG
TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC
CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG
CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC
TTGCCTCGTTCCCAAGCCCACATGTCGTTTGCATTGGTGTGGAGGTGTGCA
TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA
ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG
GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA
TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA
TGCCCACCCTCTAAAACCCAAACAAAAACAACAAAGTCAAGAAAACCCA
TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC
ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG
GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT
GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC
AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT
CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC
TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA
CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT
TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT
TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG
CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG
ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC
AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG
CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA
AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG
CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT
ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG
AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC
CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG
GCATAATTACTAGCTCATGGACTGACAAGGTCTACACTATTTCGAATCTCA
CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC
TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC
AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT
CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC
CCTCTCAGCAGCCAGGTTTTGCGTTCCATAATGTCACGTACCCCCAGTCAT
ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC
TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT
TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG
AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC
TGACTGAACATTCTTGCTGGGCCAAGTCTTAATCATCTCTAAATCCCTCTG
GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA
AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT
TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA
CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT
CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA
TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG
CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT
TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC
ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA
CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC
AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT
ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG
AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG
TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC
AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT
ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG
ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC
CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA
AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT
CCTCTGTGGCTCACTGACTTCAGAAGCCAACCTCCGTTGTTCAAGTCACTT
GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG
GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT
GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT
TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT
TGGCTTTTTTCTTGCCATTACCCTACTTGGCCAAGTGCAAATGGGATTTCA
ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA
CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC
CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC
CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC
TCCATGGGCTCTTCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCT
GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT
CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA
TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT
TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA
GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT
GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC
TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT
TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA
CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA
CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC
WO 00/46360 PCT/~JS00/02405 CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG
CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC
AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG
AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA
GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA
TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT
GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA
GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC
AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC
CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC
AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT
TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA
CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG
GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG
AAGGGGAAGGGAGGACAGAAGAGGGGAGGGAGCGGGGAGATTATCTAC
CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT
GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC
TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT
TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT
GACTTTCCCTTTGCAATGACAGGAAGATAGTATACTGCATGGGACCACCA
CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC
CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA
CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA
GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA
ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG
AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG
GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC
GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA
ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA
CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA
GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA
GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC
ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG
TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC
ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG
GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC
TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT
GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT
TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC
TCCTCCTCAGTCTTGGCCGGTCTGGGAAGAACTTGGTTTAGATGCTGGGA
ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC
CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA
CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC
TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG
GGGCCACAAATCTGCTCTGCCACTTCACTGCTGGGTGAAAGCTGTTGGCTT
GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC
TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC
CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT
CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA
AGAGTGAACAGCTGAGGAGCTCTGTGACTCTGAGAGAGAGAGAAATCAA
TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG
GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG
CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT
CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT
CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC
CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG
GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC
CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC
TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG
GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG
GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG
GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC
CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA
CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC
CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT
GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA
GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC
TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG
GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG
GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA
GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC
CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG
AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG
CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG
CTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATCAGCTGCTT
GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG
GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA
ACCGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCA
GTTCTGGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCG
GCTACACTTGGAGGTGACTTGTTCTTGGACGGCTACATACAGAAAGAGAG
AAGTGGGGATGAGTTCCAAAGGCATCCTCGACTTCGGCTGTGGCCACCGG
AGGGTAGCTCCTGGCCCAACACGGACTTCTCACCTCCCGCCCTTGGCTCTC
TACTGAGCTCCCCCCTGCTCCCCAATTCCTCGCCATTCCCCTCATTTCTCTG
CCCTCAGCCTGGACTGCAGTTCTTCTGGGAAGCTGCCCCAACTCCCTAGGT
CTGTGCTCACCAAGAGCAGATCACACTGGACTGAAATGCCAGCTGATTTG
TCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATGTCTGCTTT
TACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCCTTTGACAGGT
GGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGG
CTGGTGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGT
GACCTGGGACAGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGT
TCCCCTCTTGTCTCATAGCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTC
AGACAACATTTGTTCAACTGAACGGTAATGGGTTTCCTTTCTGAAGGCTG
AAATCTGGGAGCTGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGG
ATGGAGAGACACGGAGCAGAAGATGGAAGGTAGAGTCCCAGGTGCCTAA
GATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCACTCTAGCTGAT
ATCTACTGTGGCCAATATCTACCGGTACTTTTTTGGGGTGGACACTGAGTC
ATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAG
TCAGAGCACAAGCCCAGTGTGTCTGACCCACCCAAGAATCCATGCTCGTA
TCTACAAAAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATC
ATGGCATGTGTATGTTTTTCAACTAGGTTACAATCTGGCCTTATAAGGTTA
ACCTCCTGGAGGCCACCAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCA
ACTGGAGTGTGCCTGATGTGGCACTCCAGCCTGGACAAGTGGGACACAGA
CTCCGCTGTTATCAGGCCCAAAGATGTCTTCCATAAGACCAGAAGAGCAA
TGGTGTAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTCCTGTCTGA
GGGCAAGCAGCCTCTTCTGGCCCTCAGACAAATGCTGAGTGTTCCCAAGA
CTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGTTGCTGA
ACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACAT
CAGGCATTTTGCTAACTGCTGTATCCTAGGCCAATAAATGTTGATCACATT
TATAGCTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCT
CTGGGAGCCCCTGACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTT
TTCCATTCCCTATACCTGACAGATGGCCCAGGAACCCATTAGAAATGGAA
GGTCACTGCAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAGGATCA
GAGCCCTGGGGGTGTGGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGG
GTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAGGGGCCTGACTCAGGG
AAATCAGTCTCTTGAATTAAGTGGTGATTTTGGAGTCATTTAGACCAGGCC
TTCAATTGGGATCCTGCTCTTAGAGTTGGATGAATTATTTAACTGATTTTC
AGATCTCCTCTTTCTCAATGCTTTCAGAAGCACAGTAACTGCTTACTCTGA
AATGAATTCTCACCCCACTTCCACATATGCACCCCTTGCCCACCCCTTTGG
GAACACTGGCCTTAACTGCTTACCTTCAAATGGACTCATCTGTTGGGAGAT
ATATGCATTCTGCCGTTCAGGGGTCATTGCCATAAGACCTGATCTCTGTTC
CTCTTGCTAAACAGAAGATGAAAAAGACAAATTAGATTACAGCTACCAAT
TAATAATTAGCCTTAGGATCGCTGCGTGGGGACCTAGGACTTGGCTTTGG
TGCAGCAGAAAGCATGAATAAACACACCAGCATACACTCGCATGCATGCC
CCACCCTCTCGAGCAAAATTCCACAGGTATAAATAAAGTAAGATTCTGCA
CCTGGGTTAAAAACACAACTGCAACAGCATAGAATGGGGCAGGAGAGAC
AGAACTTAATAGCAAGAGCACACAGAAAAAAGTTTTAGGCATTTTGGATG
TCCATCTGCTCAGGATGGGTCAGCAGTGAGATGCGGTCACCAAAAGAACA
AATGTAACATTAGGCTGCATTAATAGAAGCAGAGTATGTAGAAGGAGGG
AGGTGACAGTCCTATGCTAACTCTGCCTTGGCCAGACTATACCCACAGGA
GTCTGGGCATGCCAGTCTCAGGGAGACCCAGACAGACTGGCTGCATTCAG
AGGATGGTAAGTAATGAGAGTGGGGATTGGACTTCAAACTACCCAGACA
AAGAATGGCTGAGCAAGCCAAGGATGCTGTGGCTGGGGCAGAGCAGACT
GTGGGCTATGTAGTGGTGGATACCTAGCCTCTGCAGGGCTGTCATAGGGA
AAGGACATTGAGAAGAGGACTGAGGCTTGTTCCTGGTGGTCCTGGCATGA
ACGGCCAGATGATCACATGGTCAGGTGGACACAGTCTCCAACACTGGGAG
TAGCCAAACACTTACTGCCAACCTCCCGCCCTTCTCCTGACTAGTTGCAGC
ATAGGCAATTGGGAGGAGCTTCCTGTCTCCATCTGAAAGCTGGCTGGGTG
GGCAGGGGGAGGAGCGAGCCAAGTTTCAAGGCCGCAGTTTCAGCACTCA
GTCTGGGATCGGCTCAAGGAGCAAAGGGGAAGAACATAGCCAGGAGGGA
ATAACATGAAGGCCCCCAGACCCAGAAAAGGCATGACTTGCTCTGAGACC
CTCAGCCGGTTGGTGTCAGGTTGTGACTCGGATCCAGGTCTGACTCCCAGT
CCAGTGCTTGAAGCCTCACCCCACACAGTGAGGGGAGCCCGGCCATCTCT
GCTCAACTGCTGCCATCTCTCTCCCCTTCTCAACCACCAAGGCAGCTCTGT
CTGGGAGCACAAGCTCCAAGTCCACTTTCTGGTCTGTGTCCCCCCCAAGAT
GCCAGAGGACTTGCCTCTACAACACGGGCTGCCCGTGCAGTGCCTGCTTT
TCCAGCAAAGGGCTTCTGGGAACCCTTCTCTGCACTCAGTGGGGCTGGTG
GGAGTGGGGCGGGGTAGCGACCCAGTGCTTGGGACTGTGCCCAGCTCTCA
GGCCTGGCAGCAGTTCCTGGCCTTGGTTCCTGCCAAGGCAGAGAGGACAA
ACACATGGCACCGGGAAGACTACACCAGAAGCGATTCCACCAGACTGGG
GTTTGCTTTTCTATCCCGCCCTTAGCCTGCTTCCTGTCCTGGTCCCTGCCTC
CCCCTCCACTGGAGCTGCCGTGTGGGCAGTGAGGGGCTGTTTCTCAGCTG
CCCTATGGAGCTGCCCTCTCCCTGCCAAAGCATTGGCAAGGCGGCAAGGG
GTGGGGGTGGGGATGGGGGGTGGGATCTGCCTTCTCAAGCTCTCATTATA
CTGAGCACGTCTCACCCATTATTTTATGTCATCTAGCAACACCCCATGTGG
ACACTGAGGAGCATGGGGGTCACATGACCACTGCCCAAGGCCACACCATC
CGGATCTGCCTGAGATGGTCAGGGTTGGCAGCCATTTCTGAAGGCAGTCC
TTTCGCTTTGGCTCTTCTTGTACCAGTCTCAGGACATCAGGGCAGAAGATC
TACAGTCCCCAGCTTACTGATGTGACAGCAGAGGCTCAGAGAGGTTAAAT
GACTTGCCCAAGGTGACACGGCTAAGAAGTACAGTATCTCCTAACTGCAG
ACCAGGTGCTTCTGCTGCTTCTGGGGACAGATTCCTGCGTGGCTGGCTAG
GTCTAAACGGTCCTTAACTCCATCCCCACCGGTTGCTGCATTAGTTTCATC
AAATAACACAGTTGTACAGAGGTAGGGGTTCAGGGGCAGGGGCAGATGG
AGGCTGGAGAGTGTGACTAAGGAAACAGCAGGGGAAGTGCGGTAAAGTC
CGAAGGGAGGGACGGAAAGAGAAAGCCAAGCCCAGGGGCGTGCCAGAC
AAAAGGAAAGGCCACGCCGGGGCAGGGCAGGCTTCAGCGGGTGCTGGGG
CGTCTTCATCCCGGGAAGCACACATTCCAGAGGACCCCGGAGTCTAATGG
AAAAGCTGGCCAGCCTATCACTATGGAAACTGCCAAGGCCACACAGCGCT
ACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGTGGGATGC
TTAAGTGCTGGGGTGCTGGGTGTTGGGATGCCTAGGTGCTGGGGTGCCGA
GATGCTGGAGTACTAGTGTGCTGGGATGCTGAAGTGCTGGGGTCCTGAGA
TGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGAGTGTTGGGGTG
CTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGAATACT
ATGGTGCTGGGGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGA
AGTGCTGAGATCCTGGAGTGCTGGGCTGCTGGGCCACAGGCTCTTGAATC
CATTCGTCTGCCCAGGGGAAGAAACCAGAAGATAAAGAGCTAATGAAGG
AGCTTTGGTTGAGAGGGAGGAAGTAATGGAAGGAGCAACATCTTGTGGA
GGAGCAGGAGAGAATGGACCTCAGGTTGGGAGAGAGGGCCAGGCTACAG
GCCAGAGAGGCAGAAGGATTCCAGCAGAGTGTGGGCTCCAGGAGCCAAG
GGGAAACAGGTTTCTGGGAGGAGAGAGTCCAGTACTGCTGAAGTGGCAA
GTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAGGAGGAATT
TGGAACTGTGACTAGAGTACTTAGGGGGAAGGAGGCAAGACTGGAGCCA
GATTGCTCTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCAC
TCCTTTGAAGGGTGGCAGAGAGCTGGAATTGGTGCTGGATGGGCTGTGGG
GTGACAGGGTCATGTGGAAAGCCCCTGGGGGGCACCTGGAAAAGGAGAA
GCTGACAGTACAGTGAGAGGACAGCTAAGGGAAAGCGGAATGGCAGAAC
ACGCACTGCCAGGAGGAATGAGGATAGGGTCAGGAGTGCCAGGGGCAGT
GAGGCCAGCCTGGGGTCAGGTGGCAGGACGTGTCCAGGAAGCTGGTCTG
CACTGCAGCCCACACTGGCTCAGCCTTGAGGTTCCCTGTGTGGTTGGGGT
AGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGCTCTGCGAGCC
AAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGG
GACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGG
GCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTG
GGTCCAACACAGAGCCAGAGAAGGGTGAATGGGCCTCCTGGCACCCCAC
CCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGACAGCACAGCTGAGCCC
AAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGCAGCCTGCCTGGGGCCTG
GCATGGATGGGACACTTGAGGCAGAGGGACAATAGTGGGCGCTGCAGTG
AGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCTGCCTGAGACGGGCAG
GGAGAACAGAGACAAAGTTGGTGACAGGGAATGAAAGCTGACTGAAGGA
CTTTACCCAGACCTATGAGGATATCTCTCTCAGCAGGAAGCAGGAGGGGA
CTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCT
CCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCA
CCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCCATTGAGAATTTTGGCA
GGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCGGGACATAACAGCTCT
GCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAGCCACTGATCAAATTG
GAACATTTTGTACCTTAGGGATGAGGATATCAACTCTCCCAGCCACTTAG
AGGGATAAATGTGATGATGCATTCAATTGTGACTACATCTGATCCCAACT
GTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAG
TAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCATCCATGAAACCTGGG
TACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAAGA
AGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCG
TCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGT
CTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTATAAAATGAGGGGCT
GAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAAAACTGCTGAATCT
TTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCA
CTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAATACTCCCTTGAAAA
ATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTATTACAGAACGAAGA
AAAAACACACTGGCTTGAAACAGACGCCAGATTTCAAATGTAGAGGTGA
AATACGAGGTGGCAATTAAAATGTGATTACAGAAAGTCTGGACACTGAGA
AAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATA
TACATGACGATAATTGCAGATAGAAACCATCAAAGACAAACCCCAAATC
AACTAATAATGTTTACAGATGTTCCCCCCCAAACCACAGAGCCTTACATC
AAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCTCGCCTTAACCCCA
CGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTGAATGGTAAAGGG
AGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGACAGGGCTCGGCT
CACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACTCTCCCAGCCTCC
TGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCTGCTGTA
AAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGTTCGCAAAAGAGA
TTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACATAGAAATAAATG
GCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGT
CATATCCACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTA
GCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGCATATTTGTAAC
ATTGTATAATAAACACAAAACAATGCATAGTAAACACAACTCTACTGAAA
CAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAGTATAAATACTT
CTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCAAATATGTACAC
GTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGTAGAGAGAGGG
ATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGGAGACAACTGC
GGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCT
ATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGC
CCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCTGTTCCAAAAT
GTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTGGGCTCACACA
GGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTG
ACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAAAAAGTCGGCA
GGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGACCTGGGAACTG
GCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGCAGCAGCCACA
TAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGCTGGC
CATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCT
GTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGG
TTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCC
CAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTG
AACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCCCGAAACTGCTGTGGT
CACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAACGCAGTGGGCA
TTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCAAGGACTGGGTG
CCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTCTGGTGGGGTGG
CCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCTGCCCAGGGGA
GTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCTGGAGGTGAAAA
TTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCGGCGCGC
GCGTCGACACGACAGGGACATACTGGGAATGCGCCCTTGCCGTGGAGGC
GGGGACCCGGCAGCGCTACGTATCCAGCATCAACCTGTATCCAGCATCAA
CCCGCCAAGTTCACTAACTTGGTAGGGGTGAGGTTAGGGATCCTTAGGAG
CCCAGGCAGCCAGACTTTCTGGGGAGCCCATTCCCATTTGTGTTGCCAAA
GTACCCCCAGCAGGTTGTGGGAATGTTGCCTGTGAAGAGAGTCTGTTGGG
GTGAGATCTTGTGTGTGTGCACAGGGTGACAGTTGTGTCCCATTTCCCGGG
AAGCTGTGATGGCAGCAGAACCTAGAGGAGCCTGAGAGAGTGTGGGAGA
GTGGGCCTCTGGAAGAGTAGAGGCTGCGGAGCCAGGTGCAGGGCTGTCT
GTCACCCAAAGGAAGAGGGACTGATGACTCACTGAGCGTGTGTGTCCCCT
GGTGGCAGCAGGCCCCATAGTGAACATACCATACCTTTTCTGTCCTGAGC
GATGCTCCCAGCAGTCCTGGGAGATGGAACGGTCCTTATTCGGCTCACAG
GAAGGACCGCCTTAACTGGACAGACACAGCAAGGTGCTAAAGATGCCTTC
CATCAGAGGCCAGGTTGGAAGCTCTAAAGAGACTTCTCTTGCTGTTCTCTC
ACCCACCCCCAGGTTGTGTGTGTCCCGCTGTGGATTCTCATGTCCTTTCTG
TGCCTGGTGGTCCTCTACTACATTGTGTGGTCCGTCTTGTTCTTGCGCTCTA
TGGATGTGATTGCGGACAGCGCAGGACACACATAACCATGGCCCTGAGCT
GGATGACCATCGTCGTGCCCCTTCTTACATTTGAGGTAAGCGTTCCACGGG
AAGCCTCTTCAGCCCCTGAAGCTTGCGCTTCCCCTGACAGGATTCTGCACC
CCTAGAAAGGCAGCCTCTGTCCCTCGAGCTCACAGTGAGCCCACTCCAGG
AGAGGGGAGAGAACACAGCCATCTCCGAGAGGGAGCTTCGGTGAAAGGA
GAGCATCCTTCCTTTCTCTTGGGGGCAGCACGTGGGGCTGGCAGGGAGAA
GAGTGCACCTTTTTAGCCATGGTGCCTCTGTATGGCTCCAGTTTCCACTCT
GGGGAAAGCAGAGTGGGATGTCAGATTTGTGTATTGGAGTCACGTGGAGA
ATTCTAGAATGGGAGCTGTTGACTCCTTAGAACAAACACCCGGAGGAGTT
TGCCATAAAACTGCTGGCACTGGGAACTTTTCAAGTGGATAGGCTATTGC
CGAGCTCTGAAGAGGGACATAAAAGCTCATTTCGAGCTTTCCCCAGGGAT
AGGTGGTTTCCTGCCTTTTTCTGGCGGTGCTGATGTTCCCTCTTGTGGGAG
CTCACGCGGGGGTGGGGTGGTGGGGAGGAACTGCCTAATGAAGTCTGGCT
TCCGCCTCTGCCCATTTTCGGTGCTGGCATCAACCGGGACTATGTCTCTTT
CTTTAGATTCTGCTGGTTCACAAACTGGATGGCCACAACGCCTTCTCCTGC
ATCCCGATCTTTGTCCCCCTTTGGCTCTCGTTGATCACGCTGATGGCAACC
ACATTTGGACAGAAGGGAGGAAACCACTGTATGTACTCAGCATTTCAGAA
GTCCTTGGTGTGTGTCTGGGGGGGGACCAGGGGGTGGGGGGTGGCGGATA
GAAGTCTAGGAAGGGATGAGTCCCCGAGGGCCCCAATTTAGAAGCTTGTG
TGGGAAAGTGAGGGCTGAGGAAATTCTGGGACCTTCTAAGGGAAGGGCA
TGCCGTAACTCTGGTGTTCTGCTGGCCTGCACCGGGACTTTTCTCGCAGTG
CACGCTGCCATTTGAGGTAGAACCAGACACGGCAGGCAACCTCTCAGAGA
TCCCGTTCCCTCCTCTGCAAAATGGGGATCAAGACAGATTCTTCCCAGGCC
CGGGAGGGTTTGATGGAAAATCCACATCTCCCACCCAAACCTGGGATTCA
TCCTAGGTCCCTGTTGGCCGCTCTGCCTCCCCCATATCCTTGCTGCCATCA
CCCGAGTCTTGCCTGTCTTGCCTTGCTAACACTCTATTCCCCTCCACCTGCT
TGCTGAGGCAGACACTTCCAAAACGATCTCTGCAGAGGGTGCCTTCCTGG
CAAGGCTGTGGGCTCCATGGCACGGAAGCCCAGAGCATTGCCCTTCGGAA
AGCCAGTGGGTTTGGGGGCAGGGCCTCACTGCAGCCCAGCAGCCCGGGCT
GTGCTTGCTGTTTGTGCCTCTGCCCCCTACCCCGCACCCGGGAGCAGGGA
GGGCTTGCACCGAGCTGACACTCCAGTAGCCTACAGAGAGGAGTAGTGG
GACTGGGAAAGTGGCTTTAAGGTGGCTCCATGAGTTCAGGCCCCCTCCTG
GCCAACCCGTGCATGACTACCGCCCTCACGGATTCCAGAGGGTGACAGAA
ATCTTGTTCTTGGGTGGCACTGTCATCCATGAGTTTATCCTGGCTGGAGAA
GATTAGCGGAAGACACCGTAGTCTGCGCACCACAGATATTTTGAGACTCA
CTGGAGCAGTAGTTCTCAAATTTGGGCATCCAGCAGAATCCCAAAAGGGC
CAGGAAAAGGGGACCGCTGGAGCCCACCCTAGCCCGACTCAGTTTCTGGA
GGTCTGGGCTGGGGCCCGAGAATGGCATCCCTAACTAGGCCCCGTGGACG
CTGTCCCTGCCGGTCCGGGAACCCCACTCCAAGCACCACAGAGCTAGCAT
TTGCACTTCTTCCCCATTTTGGGTACTCAAGCCCTGTTCAGGCTTTGTGACT
CAGGAGTCTGGATAAAGTATGTTATGACATTGTAGGAGTGAAACTTCTTG
TTACGGAAAGAAAGTTAACAGGAAGGTCAGTTGAGCCTCGTGTGTGAAAT
AAAAAATTCTTATTTTTCAGGGTGGTTTGGTATCCGCAAAGATTTCTGTCA
GTTTCTGCTTGAAATCTTCCCATTTCTACGAGAATATGGAAACATTTCCTA
TGATCTCCATCACGAAGATAATGAAGAAACCGAAGAGACCCCAGTTCCGG
AGCCCCCTAAAATCGCACCCATGTTTCGAAAGAAGGCCAGGGTGGTCATT
ACCCAGAGCCCTGGGAAGTATGTGCTCCCACCTCCCAAATTAAATATCGA
AATGCCAGATTAGATGCCACTTCCGGGGACAGAGCTTAAGTGGACTGGGA
CGCACTCTCTCCGCCTTCCTCTGCCCCCTCGTTCACCCCGCAGACCAGAAC
CAGTACTGGAGCTGGGTCTCCAGGTACGTCCATCTCATGCCTTGTTTGCAT
CCAGCGCCTATCAGCCACTCACCACGACGGGACGCGGAAGTGGCAGGTG
ACGGGGGTGTGTGCCAGCAGATGCGGATGCCAGGAAGAGTGTGAGAACA
GGGGTGGGATTACCGTCTGTCTGGGAGGGGCTCCAGGTACCCCTCTTCCC
CGTCAGACCCACTGGGAGATGGCTGCTTGCCAGGCCCCCAGAAGGAACAT
CTGTCTATACGGTGCTGAAATCCCAATCAAAAGTATTGTTTAGAAATGTAT
TTCTCCACAGGGCTGACCTCCTGCAGCTCGCTGAGCACTCCCAGGTCCTCA
GCACTCCCAGGTCGTGGCTGGGGCAGTCAGTAGGAACTGTAACTATGTCT
CTGATGCACCACGTGTTTAGACACAGCACAGTCCTTTTTTCTGTTCCTACT
GTGGAAGTAGTTTCTCTTTGGGCATGCTGACAGCAGTTTTTCATAGCCTCA
CGGATGAGCCCTTTCTACGGGAGTGACTCCATGCTTGTATACAGAGTATTT
ATACAAATGTTTTAGCATCTTCATATGCGGTGTTAACCCCTAGTTCTGTAC
AGCATATTCTGTTCAAGTATTTTTTTACAAGCTTGTGCTGTAGGCACATGC
CTTCTGCTGCAGAAGTGGACGCCCGTGGCACACTCCCCCCCCCCCCCCGT
GGGGTGCCACGCCTTCATGGGACATTGCCACTTCTGCCCTGGAACTCGTG
CAGGTACGTAGTAGCTGCTACTGCCACAACGGCAACACCAAGCAAGAGA
TGGTCCATGCTTTTCTGACGTTCTCAGAATAGTGGCTAGCTTCAAACCTGA
CAAGCGCTGCTTGAAGCCGGAACACTAGAGAATGTTGCTGAGAGCAGAA
ACGGCCACGCGGGTCACGACTATGCGTGGGAAAGTCTCAAGCTTCCCTCC
TGCCAGCAACAAGAAGGCTTTGGAGTAGGCATGATGTTTTCACGTGTGCG
TGCCGTTTCTCCAAGCACTGCAGGTTCCACCGTGTGTCAGAGGCTGCAAG
TTTAACATCCTCCTGCCTGAAAACAAATAGGTCCTTTGCTGAAAAGAGGG
TAAAAAAAGAGCTTTGATCTTCTCAGCCAGGAGAAGAGGGTGGTGTTTTC
ACGCGGGCAACTGCTCGCCGGCCTACATGGGGTTAATTCAAGTCTGCTGC
GAGCACGACTCCGCCCTTGGCACTGGCCTCCAGCAAGCCCTGTTCTCTTTG
GGGTACAGGGGAACGGGATGGTTTAGACTTTCCTGCTCAGTGTGTAAAAA
ATGTAGCTAAAGCCACTATTTTTGCTCTCCTTAAGCTGTTCAATAAACCGG
TTCCTCATTTTACACGTGCATGATGTGTATCTTCTTTGCTGGATGGGCCAG
GAAACTGGAGTGGTCCTCTCAGCCAGCCTCAGAGGAAAGAAATCTCTAGC
TGGCACAGGCAGCCAGTGAGTGAGGCTGGCGGCTGCAGGGGCACAGCCT
TTAGAATGAGTCCTTCAGTGCACAGGTCCCAGGGTATACGGGGTAGTGGG
AGGAAGGAGGGGACGCCTCGCAGATGCCACTGTTGGCTGGGCTACACCTT
GCCACACTTGTTACTGCTTAGGAGGCTTTCTGGAGTGTTCCTTGGGTGCTA
CGACAATCTGCAGCAGACACTGTCCTTTCACCGCTCCTGGTCCTCGTTTGC
TCCCCAGTGATGTCAACAGCTGAGGACTGCTCACGCTGCAACAAAAGGCT
CTGCAGTCGCTGTCTAGCTTGCCCTAGTCGTCTCTAGAGTTCTGCCTGAAC
TGAAACTCAAGTGGGGTTCAGCTCATGACTTGTGGCAATTGACCAGGAAA
TTCACCAGTTGCTGTGGCTGGAAGGATTTTCAGTCCTGTGGGTTGTAACCA
GAGGCCACAGGTGGATTCTGCCTTAGGCTCATGAGATTTCCGACTTGCTGT
TGAAGAAAATGCCTTGTGAAGTGACAACAGTAGCTCTGACCCAACTGCCG
GTGCCTCGCTAGTTCCTATACGTCCCACTGGATCCTCACAGCCCCGGGAA
GCAGGTGCTACTACTCTTATCCCCGGGAGGAGACAGAGGCCGAGAGAGG
TTAAGTGACGTGCCCAAGTCACACAGCTCGGCAGCGGCCGGGTTGAGCAT
CAGCAGTCTGTTTGCAGACCCCTCACTGTCACCCCCTGAGCCAGTGCGCCT
TGGGCCCTGCGGTCAGGATGTCTCAAGCGTGGAGGCATCACCGGTTCGTG
GCAGTCTCTGGAAGGTCACTGAGCTCTGTGCCCAGAATCGAGTCGGGGGA
GTCTGTGCAGAGGTGGCCCTGTGTGTGGGGACAGTGTGTGACACAGACAC
TGCTTTGGATGGACACCTCTCCCGTGACCTCCTAGCATCCAATCCCAAAGG
AACAACTGTTGCAGAGATGGACCGCTGGACACAAACCCACGTGCGTTTCT
CTGGAGACACTGGCCAAGGAAAACAAAACATGCTCGAAGGCCAACAGCT
GCATGCCCCACCGCGATGTGACCGCAGACACCCGGGGTGTAGAAGGGTCT
CTGCCTGGTGGGGGGACACGTGCAGGCCGAGGAGAGGCAGGAAGGAGGC
TGCCTCCGACTCCCCACTGGACTGCATGGCGACGGCGTGTGGTGGGGCAG
TCAGCTAAGCCATTTGCCTAAGGGGCTGTCGGGCATCTGCGTGCTGGGGA
CCGACAGTGTGGGTGTGTTAGGAGGATCTGTATGGAGCACATTGCTGCCT
CTGGCTAGGACAGGGTGGAAAGGGTGGCGTGGCTACAGCCTGACCCATG
GGCACCGTCCTACCCTTTGTTCTGTGCTTCCGAGTGTCAGTCATGTGCTGG
GGTCTGTGGGCCCATGACTCAGACGGTGAGCTCTGACCTTCCTGAGCCAG
GGCTTTGCTGTAGTTGTGCCTGGCTCAGGAGCTCTAGGACAAGGGGACCG
CTCCAGGTCTGCATCTACGGTGTGGCAGGGCCCCTCGGCACTCTTGTGCAC
TAGTGTCATCTTTCCCATTGAAATGACTGTGAGGACCAGAATGTGCACAT
GCAGATGGGCAGCTACTTGTCTGCCTTGGCCCTTTATTACACAACTTGCTG
GGGGTGGAGATGCCACCCCCCGGCAGTCAGAGCCCCTTTATGATGTCATG
GGGCTGGTTACATGACTGCCAAGGGGTGCTGCTGGCCACACTGCACTAGC
AAGTTTGCCAGATGGAGGACAAGCGATCATTGAGTATGGCTCGCTGTGAA
GAAAGAAATTCGAGAGGACAGGATCATGGCTTGGAAAGGGTGCCTTTCCC
TCCCCAGTTGCAGTCAGAGACCTACCTTCACCCAGCAGATCCTTCCCCTGC
CTGGGACGACCCGGGGTCCACTGGGAGCCCTAACTTGAGGCTGCTGACAG
AAGAAATCGCTTTCCAACCTCTGGCCGAGGAAGCTTCGTTCAGAAGGCCG
CACCCTGACGGTGACGTCCCGCCCCAGGGAGAAGATAATCTCCTCTCCCT
CCCCTTTCCACAGAAACTGTGGAGACTGGTCAGCAGCAACCAGTTTTCGT
CCATCTGGTGGGATGACAGTGGGGCTTGTAGAGTGATCAATCAAAAACTC
TTTGAAAAGGAGATTCTCAAAAGGGACGTCGCACACAAAGTGTTTGCCAC
AACTTCGATAAAGAGCTTCTTCCGCCAGCTAAACTTGTATGGCTTCCGAA
AACGGCGTCAATGCACTTTCAGGACCTTCACCCGCATTTTCTCCGCAAAA
AGGCTGGTCTCCATCTTGAATAAGGTAATGAACGACAAGCCTCTGGAGGG
GTTAAGTCGGTGGGCTCTGGGGCCTGGTCGGGTGGAAGTCCCAGGACTGC
CTCCTGGGAAGTGGGCGACCTCAGGCAGGGTGTGGGGCCATCGCTGTGGG
CCTGTGTCCCCCTCTGGGTGGAGGTGACATGAACTAAGAGTGAATGTGGG
GAGAGGGCTGAGGATGGTGCGGGCCCCTCTCGAGTGTGTAAAATATCACA
GGTGCCAAGTAGCCGTATCTGCGTGTCGTCCTCCCCGGGGCCAGCCATGT
CATCTGGTGGTTGCTGTGTCCCCCTGACTCCACAGCACATTACCCTGTGAG
GTGAGCAGGCCAGGGGAGTCTGGTAT'T"TGTACCACTGTCACCCTAGCTGG
TGTCTGGAGAGGTGCTCAAGTGGAAGCACTGAAGGGCGCCTGGCGCAGG
AGGTGCAGATGCTCCTGCTGCCCTTGGTAGGTGGGCCCCTGGTGTGGAAG
AGCCAGTACCCAGGGCCTCCAACCCAGCCGGGGTGCATTCTGTTGCCAGC
TGACACTGCATGGGGGAGGCCCAGAATCTTCTTCCCTCCTGGTCTGCAACT
TCAAAGACCCTTTCCGCCGGCCATGGACACCCTAATCTGCCATTTTGAGGC
TTTTTCCAAGACGGAAAGGCCCGCCACAACTTGGTAAACCTTGACGATGT
GAACGCGAGTCCCCAGCTTCCTTTGGGGACTGGGACCTTTTCCAGAAAGG
CCTCCTGGGCCAGTAGAGTTCTCTTGCACAGGGGCGTAGATGGTTGGTAG
TTGTAGTCCATCCTTGTGACTTGCAGTTTCCTTTTGTTTATTTAAACTTTGA
CTTATAGTTAGAGTTCTACTGCCATCCTTACTTTCAAAGAGACTCCCCTCA
CCTCCTCGTGAGGATGAAGAGAAGAGTGGGTGTCAAGTCTGCACCAAGAC
ATCAGGAGGAGGACAAGCCAGAAGCTGCTGGATCCTGTCTGGCACCAGC
AGACACTGAGCAACAAGATCACACGTCTCCGAATGAGAATGACCAGGTC
ACACCGCAACACCGGGAACCGGCCGGTCCCAACACCCAAATCAGGAGTG
GCTCTGCTCCACCAGCAACTCCTGTGATGGTGCCTGATTCCGCCGTGGCGA
GTGACAACAGTCCAGTGACCCAGCCGGCCGGCGAGTGGTCAGAGGGCAG
CCAGGCTCACGTCACTCCGGTGGCCGCTGTCCCTGGGCCTGCAGCGCTGC
CCTTCCTCTATGTCCCTGGATCTCCCACTCAGATGAATTCTTACGGGCCTG
TGGTGGCCCTTCCCACAGCGTCCCGTAGTACCCTTGCCATGGACACCACA
GGACTTCCTGCACCTGGCATGCTGCCCTTTTGCCATCTCTGGGTACCGGTG
ACCCTAGTGGCTGCTGGGGCTGCACAGCCTGCTGCCTCCATGGTCATGTTC
CCCCATCTCCCAGCTCTGCACCACCATTGCCCCCACAGCCACCGCACGTC
ACAGTACATGCCAGCTAGCGATGGGCCCCAGGCGTACCCAGACTACGCAG
ACCAGAGCACATAGAGGGCAGCATTTGGGCAGAATATGTGCTGGTCAATA
AATGTGTCAGAAAATGAGTAATTTTCTGACTGCACAAAAAAGTCTTCATG
GTCTCCATCCTTTTTCTTTCTTTGCAGTCCAGTACCCCCCTCCGACATCTTT
GGCTTGCATGATAGAGACTTCAGCCTAACACGCCTCCCTGGAAAAGTGCC
CAGGAGATACTCGAGCAGGTGCTGGGTTTTAGGAAGCCCCTCAGACATCC
CGCCTCTACTCATTAAAAATTACCTGGAGCTGGCCGGGCGCAGTGGCTCA
TGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGAGCT
CGGGAGATCGAGACTGTCCTGGCTAACACGGTGAAACCCCGTCTGTACTA
AAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCACCTGTAGTCCCAG
CTACTCGGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGGAGGCGGAC
GTGCAGCAAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAG
CAGGACTCCGTCTAAAAAAAAAAAAAAAAAAAAAAAATTACCTGGAGTT
GCTCGCTGAGTGCTAATTGTGCTGCTAGCATTGCCGGCCTAGGGTTCACA
ATGACTCTGTGTTACTGGCCATTCCTTGTCATGGCGGTGCCTTTATATAAC
AGGATCTTCACTTTACATACAGATGAGATCACGTCTGGCTTTGTCATTGGG
TTAAAAAGTGCCCTTGATAGTTGTGGAGGTGACACATACTACCGTCGACT
CGAATAGATCTGCTAGGCGCCAAGCTTGTCAGCCTTACCAGTAAAAAAGA
AAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCAC
AGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAA
TGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGA
ACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGT
CACTTCCGTTTTCCCACGTTACGTAACTTCCCATTTTAAGAAAACTACAAT
TCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCG
TTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG
GCTTCAATCCAAAATAAGGTATATTATTGATGATGTTT
SEQ. ~. NO. 17 Helper dependent vector C4AF0 The sequence is shown without transgenes.
AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG
AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG
ACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATG
TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA
CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT
GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA
GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC
TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT
TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC
GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC
CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT
ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC
TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG
GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG
CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG
AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA
GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA
AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC
CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT
CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC
ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA
TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC
CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA
GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA
AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC
TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA
ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC
TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT
ATTTTGATGTAATATTTGAAATAACAATAAAAATTGCCACCACTGAATTTA
TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG
TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC
CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG
CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC
TTGCCTCGTTCCCAAGCCCACATGTCGTTTGCATTGGTGTGGAGGTGTGCA
TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA
ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG
GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA
TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA
TGCCCACCCTCTAAAACCCAAACAAAAACAACAAAGTCAAGAAAACCCA
TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC
ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG
GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT
GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC
AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT
CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC
TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA
CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT
TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT
TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG
CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG
ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC
AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG
CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA
AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG
CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT
ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG
AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC
CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG
GCATAATTACTAGCTCATGGACTGACAAGGTCTACACTATTTCGAATCTCA
CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC
TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC
AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT
CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC
CCTCTCAGCAGCCAGGTTTTGCGTTCCATAATGTCACGTACCCCCAGTCAT
ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC
TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT
TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG
AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC
TGACTGAACATTCTTGCTGGGCCAAGTCTTAATCATCTCTAAATCCCTCTG
GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA
AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT
TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA
CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT
CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA
TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG
CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT
TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC
ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA
CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC
AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT
ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG
AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG
TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC
AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT
ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG
ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC
CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA
AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT
CCTCTGTGGCTCACTGACTTCAGAAGCCAACC~'CCGTTGTTCAAGTCACTT
GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG
GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT
GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT
TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT
TGGCTTTTTTCTTGCCATTACCCTACTTGGCCAAGTGCAAATGGGATTTCA
ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA
CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC
CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC
CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC
TCCATGGGCTCTTCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCT
GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT
CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA
TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT
TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA
GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT
GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC
TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT
TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA
CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA
CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC
CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG
CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC
AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG
AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA
GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA
TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT
GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA
GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC
AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC
CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC
AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT
TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA
CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG
GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG
AAGGGGAAGGGAGGACAGAAGAGGGGAGGGAGCGGGGAGATTATCTAC
CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT
GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC
TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT
TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT
GACTTTCCCTTTGCAATGACAGGAAGATAGTATACTGCATGGGACCACCA
CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC
CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA
CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA
GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA
ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG
AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG
GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC
GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA
ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA
CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA
GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA
GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC
ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG
TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC
ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG
GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC
TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT
GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT
TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC
TCCTCCTCAGTCTTGGCCGGTCTGGGAAGAACTTGGTTTAGATGCTGGGA
ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC
CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA
CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC
TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG
GGGCCACAAATCTGCTCTGCCACTTCACTGCTGGGTGAAAGCTGTTGGCTT
GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC
TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC
CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT
CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA
AGAGTGAACAGCTGAGGAGCTCTGTGACTCTGAGAGAGAGAGAAATCAA
TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG
GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG
CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT
CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT
CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC
CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG
GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC
CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC
TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG
GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG
GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG
GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC
CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA
CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC
CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT
GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA
GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC
TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG
GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG
GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA
GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC
CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG
AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG
CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG
CTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATCAGCTGCTT
GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG
GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA
ACCGCGGCCGATCTGAATTCAGATCGGCCGCGGGATCATTCCTGGACTCA
GATTGTTCTGAAGAAGCCCAGTTCTGGGTGGCATCAAGTGCTTGCTAGAT
GGGGGGCTTGCCTTGATCCGGCTACACTTGGAGGTGACTTGTTCTTGGAC
GGCTACATACAGAAAGAGAGAAGTGGGGATGAGTTCCAAAGGCATCCTC
GACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACGGACTTC
TCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTCC
TCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGG
GAAGCTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAGCAGATCACACT
GGACTGAAATGCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTG
GAGGTCAGGGTCCATGTCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCT
GGCACTGCCCACCCTTTGACAGGTGGTGCATATTTTGTAGAAGGAAGGAA
GGGGCCAGGTGGGGTGGGCTGGGCTGGTGGCGGGAGCTAGCTCAGCCTCT
TAGATTCTCTACCCGATGGATGTGACCTGGGACAGCAAGTGAGTGTGGTG
AGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGC
CTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGAACGGTA
ATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTCC
CTGAGTTCTGAAGAGCCTGGGGATGGAGAGACACGGAGCAGAAGATGGA
AGGTAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTC
ATGAGAGTCCACTCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACT
TTTTTGGGGTGGACACTGAGTCATGCAGCAGTCTTATGGTTTACCCAAGGT
CAGGTAGGGGAGACAGTGCAGTCAGAGCACAAGCCCAGTGTGTCTGACC
CACCCAAGAATCCATGCTCGTATCTACAAAAATGATTTTTTCTCTTGTAAT
GGTGCCTAGGTTCTTTTATTATCATGGCATGTGTATGTTTTTCAACTAGGTT
ACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCACCAGCCTTCCTGA
AACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGGCACTCCA
GCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGTC
TTCCATAAGACCAGAAGAGCAATGGTGTAGAGGTGTCATGGGCTACAATA
AAGATGCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCTGGCCCTCAGA
CAAATGCTGAGTGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCC
ACTGGTGCGTAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAAC
CTCCACGGAGTGGGAACTACATCAGGCATTTTGCTAACTGCTGTATCCTA
GGCCAATAAATGTTGATCACATTTATAGCTGCCATGGTAGGGTGGGGACC
CCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCCTGACACAAACTTTCTGA
AGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCC
AGGAACCCATTAGAAATGGAAGGTCACTGCAGCAGTATGTGAATGTGCGT
GTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGT
GATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGC
TTTAAGGGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAAGTGGTGATTT
TGGAGTCATTTAGACCAGGCCTTCAATTGGGATCCTGCTCTTAGAGTTGGA
TGAATTATTTAACTGATTTTCAGATCTCCTCTTTCTCAATGCTTTCAGAAGC
ACAGTAACTGCTTACTCTGAAATGAATTCTCACCCCACTTCCACATATGCA
CCCCTTGCCCACCCCTTTGGGAACACTGGCCTTAACTGCTTACCTTCAAAT
GGACTCATCTGTTGGGAGATATATGCATTCTGCCGTTCAGGGGTCATTGCC
ATAAGACCTGATCTCTGTTCCTCTTGCTAAACAGAAGATGAAAAAGACAA
ATTAGATTACAGCTACCAATTAATAATTAGCCTTAGGATCGCTGCGTGGG
GACCTAGGACTTGGCTTTGGTGCAGCAGAAAGCATGAATAAACACACCAG
CATACACTCGCATGCATGCCCCACCCTCTCGAGCAAAATTCCACAGGTAT
AAATAAAGTAAGATTCTGCACCTGGGTTAAAAACACAACTGCAACAGCAT
AGAATGGGGCAGGAGAGACAGAACTTAATAGCAAGAGCACACAGAAAAA
AGTTTTAGGCATTTTGGATGTCCATCTGCTCAGGATGGGTCAGCAGTGAG
ATGCGGTCACCAAAAGAACAAATGTAACATTAGGCTGCATTAATAGAAGC
AGAGTATGTAGAAGGAGGGAGGTGACAGTCCTATGCTAACTCTGCCTTGG
CCAGACTATACCCACAGGAGTCTGGGCATGCCAGTCTCAGGGAGACCCAG
ACAGACTGGCTGCATTCAGAGGATGGTAAGTAATGAGAGTGGGGATTGG
ACTTCAAACTACCCAGACAAAGAATGGCTGAGCAAGCCAAGGATGCTGT
GGCTGGGGCAGAGCAGACTGTGGGCTATGTAGTGGTGGATACCTAGCCTC
TGCAGGGCTGTCATAGGGAAAGGACATTGAGAAGAGGACTGAGGCTTGTT
CCTGGTGGTCCTGGCATGAACGGCCAGATGATCACATGGTCAGGTGGACA
CAGTCTCCAACACTGGGAGTAGCCAAACACTTACTGCCAACCTCCCGCCC
TTCTCCTGACTAGTTGCAGCATAGGCAATTGGGAGGAGCTTCCTGTCTCCA
TCTGAAAGCTGGCTGGGTGGGCAGGGGGAGGAGCGAGCCAAGTTTCAAG
GCCGCAGTTTCAGCACTCAGTCTGGGATCGGCTCAAGGAGCAAAGGGGA
AGAACATAGCCAGGAGGGAATAACATGAAGGCCCCCAGACCCAGAAAAG
GCATGACTTGCTCTGAGACCCTCAGCCGGTTGGTGTCAGGTTGTGACTCG
GATCCAGGTCTGACTCCCAGTCCAGTGCTTGAAGCCTCACCCCACACAGT
GAGGGGAGCCCGGCCATCTCTGCTCAACTGCTGCCATCTCTCTCCCCTTCT
CAACCACCAAGGCAGCTCTGTCTGGGAGCACAAGCTCCAAGTCCACTTTC
TGGTCTGTGTCCCCCCCAAGATGCCAGAGGACTTGCCTCTACAACACGGG
CTGCCCGTGCAGTGCCTGCTTTTCCAGCAAAGGGCTTCTGGGAACCCTTCT
CTGCACTCAGTGGGGCTGGTGGGAGTGGGGCGGGGTAGCGACCCAGTGCT
TGGGACTGTGCCCAGCTCTCAGGCCTGGCAGCAGTTCCTGGCCTTGGTTCC
TGCCAAGGCAGAGAGGACAAACACATGGCACCGGGAAGACTACACCAGA
AGCGATTCCACCAGACTGGGGTTTGCTTTTCTATCCCGCCCTTAGCCTGCT
TCCTGTCCTGGTCCCTGCCTCCCCCTCCACTGGAGCTGCCGTGTGGGCAGT
GAGGGGCTGTTTCTCAGCTGCCCTATGGAGCTGCCCTCTCCCTGCCAAAGC
ATTGGCAAGGCGGCAAGGGGTGGGGGTGGGGATGGGGGGTGGGATCTGC
CTTCTCAAGCTCTCATTATACTGAGCACGTCTCACCCATTATTTTATGTCAT
CTAGCAACACCCCATGTGGACACTGAGGAGCATGGGGGTCACATGACCAC
TGCCCAAGGCCACACCATCCGGATCTGCCTGAGATGGTCAGGGTTGGCAG
CCATTTCTGAAGGCAGTCCTTTCGCTTTGGCTCTTCTTGTACCAGTCTCAG
GACATCAGGGCAGAAGATCTACAGTCCCCAGCTTACTGATGTGACAGCAG
AGGCTCAGAGAGGTTAAATGACTTGCCCAAGGTGACACGGCTAAGAAGT
ACAGTATCTCCTAACTGCAGACCAGGTGCTTCTGCTGCTTCTGGGGACAG
ATTCCTGCGTGGCTGGCTAGGTCTAAACGGTCCTTAACTCCATCCCCACCG
GTTGCTGCATTAGTTTCATCAAATAACACAGTTGTACAGAGGTAGGGGTT
CAGGGGCAGGGGCAGATGGAGGCTGGAGAGTGTGACTAAGGAAACAGCA
GGGGAAGTGCGGTAAAGTCCGAAGGGAGGGACGGAAAGAGAAAGCCAA
GCCCAGGGGCGTGCCAGACAAAAGGAAAGGCCACGCCGGGGCAGGGCAG
GCTTCAGCGGGTGCTGGGGCGTCTTCATCCCGGGAAGCACACATTCCAGA
GGACCCCGGAGTCTAATGGAAAAGCTGGCCAGCCTATCACTATGGAAACT
GCCAAGGCCACACAGCGCTACGCGTATTTAAATGTGCTGGAGTGTTGAGA
TACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGTGTTGGGATGC
CTAGGTGCTGGGGTGCCGAGATGCTGGAGTACTAGTGTGCTGGGATGCTG
AAGTGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGG
CTGCTGGAGTGTTGGGGTGCTGGGATGCTGGAGTGCTGAGATGCTTGGAC
AATGGGGTGCTGGAATACTATGGTGCTGGGGTGCTGGGGTATTGAGATGC
TAGGGTACTGGGATGCTGAAGTGCTGAGATCCTGGAGTGCTGGGCTGCTG
GGCCACAGGCTCTTGAATCCATTCGTCTGCCCAGGGGAAGAAACCAGAAG
ATAAAGAGCTAATGAAGGAGCTTTGGTTGAGAGGGAGGAAGTAATGGAA
GGAGCAACATCTTGTGGAGGAGCAGGAGAGAATGGACCTCAGGTTGGGA
GAGAGGGCCAGGCTACAGGCCAGAGAGGCAGAAGGATTCCAGCAGAGTG
TGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTCCA
GTACTGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCT
TTATGACCAGGAGGAATTTGGAACTGTGACTAGAGTACTTAGGGGGAAGG
AGGCAAGACTGGAGCCAGATTGCTCTGGGTTGAGGGGTGAGTGGGAGGT
GAAGCAGGGCACTGTCACTCCTTTGAAGGGTGGCAGAGAGCTGGAATTGG
TGCTGGATGGGCTGTGGGGTGACAGGGTCATGTGGAAAGCCCCTGGGGGG
CACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGAGGACAGCTAAGGGA
AAGCGGAATGGCAGAACACGCACTGCCAGGAGGAATGAGGATAGGGTCA
GGAGTGCCAGGGGCAGTGAGGCCAGCCTGGGGTCAGGTGGCAGGACGTG
TCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCTTGAGGTT
CCCTGTGTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGA
ACCAGCTCTGCGAGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTG
GCAGAGCTGAGTGGGGACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGT
GGCCCCTCCGGGAGGGCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCA
GTGGCTGGGCAGGTGGGTCCAACACAGAGCCAGAGAAGGGTGAATGGGC
CTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGAC
AGCACAGCTGAGCCCAAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGCAG
CCTGCCTGGGGCCTGGCATGGATGGGACACTTGAGGCAGAGGGACAATA
GTGGGCGCTGCAGTGAGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCT
GCCTGAGACGGGCAGGGAGAACAGAGACAAAGTTGGTGACAGGGAATGA
AAGCTGACTGAAGGACTTTACCCAGACCTATGAGGATATCTCTCTCAGCA
GGAAGCAGGAGGGGACTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGG
GAAAATGACTCTTTCTCCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTG
CGGAGGTCTGCTTCCACCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCC
ATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCG
GGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAG
CCACTGATCAAATTGGAACATTTTGTACCTTAGGGATGAGGATATCAACT
CTCCCAGCCACTTAGAGGGATAAATGTGATGATGCATTCAATTGTGACTA
CATCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGA
GGCGTGCATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCAT
CCATGAAACCTGGGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAG
GGTCTGGTCAAAGAAGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGAC
GGTGGTGCAGAGCGTCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCT
CCCGGGGCAGTTGTCTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTAT
AAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAA
AACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAA
AGAACCAAAGCCACTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAA
TACTCCCTTGAAAAATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTAT
TACAGAACGAAGAAAAAACACACTGGCTTGAAACAGACGCCAGATTTCA
AATGTAGAGGTGAAATACGAGGTGGCAATTAAAATGTGATTACAGAAAG
TCTGGACACTGAGAAAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAA
CAGACACTTAAATATACATGACGATAATTGCAGATAGAAACCATCAAAGA
CAAACCCCAAATCAACTAATAATGTTTACAGATGTTCCCCCCCAAACCAC
AGAGCCTTACATCAAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCT
CGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTG
AATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGA
CAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAAC
TCTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAAC
AGCTTCTGCTGTAAAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGT
TCGCAAAAGAGATTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACA
TAGAAATAAATGGCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACA
ACAACTCTTGGTCATATCCACATTGCTCATTGCATTAAAACCATAAGCGAC
TCAGCCACCTAGCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGC
ATATTTGTAACATTGTATAATAAACACAAAACAATGCATAGTAAACACAA
CTCTACTGAAACAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAG
TATAAATACTTCTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCA
AATATGTACACGTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGT
AGAGAGAGGGATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGG
AGACAACTGCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCAT
GGGGGTTTCTATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTG
GGTGACTTGCCCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCT
GTTCCAAAATGTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTG
GGCTCACACAGGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTG
CAGTCAGGTGACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAA
AAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGAC
CTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGC
AGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGAC
TAAAGCTGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGC
ACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCT
GAAGCTGGGGTTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCA
CTTGTGCCTCCCAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGT
GAGTGCTGCTGAACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCCCGAA
ACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAA
CGCAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCA
AGGACTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTC
TGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTC
TGCCCAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCT
GGAGGTGAAAATTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCA
TGCCCGGCGCGCGCGTCGACGTGAGCAAGTGGAATGGAGTGGCAGGGAA
TGAGTGAGGGAGACAACAGCTCAGTAGGACCTTGTGGGCTACTGGAAAG
ACTTTGGCTTTGACTCTCAGTGAGATAAGGAGGTATTTGAAGGAGTTTTAG
CAGAGAAGGAACAATTCTGTTTATTTTTAGGAGCTCTTTATATATTTAGGA
AATTACTTCTTTGTTAGTCATATATTTTGGAATTTGTTGTTCCCCGTTTGTC
ATTTCTGTTTCTACATTTTGCTTATAATATTTGTCTATGTAAACCTTAATAT
GTACTTGGTTAAATTGATAAATTCTTTGCATAATTAATAGCTAGCTATTGT
TGGCCTACCTATAATTAATTTTTGCTTTATCTAGTAATAGGCCTGAATTTTC
ATTTGGGATCCAGCCCTCTCCCTGACACTATGCAGGGCCTGGGAGACTAA
AGCCTGCTGGAAATAACTCCAGCTTGGCCCTGCCAAACATTACAACACCT
AGCACCTGCTCTCCCCACCATAGTGACTGATTTGGGCAGGAATGAGACCA
CAGCCACTCCAAGAGCCCAGCTGTTGGCTACCAGTAACATCTTCTCCTCTA
AACAGGAGATCCAGCTATATTGAAGGCTTCCAAAATTACCCTATGTCTAC
CTGTCACAGGAATGTTCAGAGGACTAAGCCTCTGCCTTTCTCCACCCCATA
CTTACTTCATATGGAACTATGAATGGGCAACTTCACAATTTATTATTTACG
TCACCTAGATGCAAGTCAGAGGGAGAAGCAACAAATCAGCAGTACCTAA
GAAGTTCTCAAGTCCTCAAGGTGAACTTTACCTATATATACAAAATATATT
TGCCTCTTTCCTTTAACCCTTTTTCTTACTCCCACCACTTTCCACCCAATGT
GTAATAATATTAGTCACTTCATAGAGTTCTTATGAGAAGTTAAATGAGGA
AATCATGTAAACCACTGAGGGCTGGCAACAAGTTCTTAATAAGTGCTATT
GAGTTATGAGTATTATTATCTCCCTTGGGCTGGATTCACAGTGTCACCTCT
TCTGGGACCATAGTAAGCCAATGGTTCCAGCTCCTTCTATTATGTCATTTT
AATTTGGGGAATAGCTTAACATATTAGATGATCCTTTACATGCCTGAGCG
GTCTTTCTTTGATGGTATTTCTATGATCTTATGAAGAGCTAGATGTTGCCG
CTCCATTTTATCTTGCTGGTGGCAGCTGGTGTTTAGCCAACTTGTCTTTTTT
TTTCTGTCTTTATATTTTTGGTTTTGTTTTTTGGCAGAAGCCTGTGTTTTTCA
GATTACTTTTTGCTTGTGTCTACTTTTTCCCAAAACAATATTACATATTAAA
CTTATTGTATATTTAAGTTTATTTTATGCATCAAAAGTCGCATTGAATTTCT
GTGCTATTGTGGGGCAAATTGACATCAATGTAATTTAACTCATTTTATCCC
AAATTAGGCTATGTCTTTCCATATACTCAAATCTTCTTTTATGTCCCTCAAC
AGTTTCCTTCATATAAATATTGCATATTTCTAATTAAGTTTATTTTTCTAAT
CTCCATTTTGGATGTCTTAAATATAGTCTTTTCTCCCTTTGCTCTTTATCAA
AGATCTGCAAACTATGGCCTGTTTTT'GTAAATAAAGTTTTGTAATAAAGTT
TAAATGGCCACTCCCATTTGTTTACATGTTGAGTATGGCTGCTTTCAGACT
GAAATGGCGAATGAAGTGGTCATAACAAGACCATATGGCCTGGAAAGCA
AATATATTTATTATCTGGCAATTTACAAAAAAAGTTTGCCAACCTCTGCTT
TATATTTTTGATCTAGTTGTTATTTGAAAATAAAGCTATTGATATTTGTATA
TTAATTTTTTACCCAGCTACCTTATTGTTTTTATTACTTAATAGTTTACAGT
ATTTCTTTTAGTTTTTCCAGGTGTAACATCTGTAAATATTAATAAATCTATT
AGCTAATATTTCATTTAGCATTTTTATATCTATACTCACATGAGAAGTTTT
CTTTAATTTTCTTTTGTGTTTATCAACCTGTGTTCACTCAGGAAAATGAAG
CCACTTAAGTATACCAGGAGTGGAGGGTTTCATATAGAAATGAGGGCTTA
TTTGAATTTTGGGAAAGCTGATGAAACAGGTCAGCAGAGTTACTGCAGAA
GACCAGGAAAGTTACATCTGGAAGGTTAGGGAAGCAGACACTAAAGACT
TAAGCCTGAAGCATAGAAGTAAGGATCCGTCAATACTTATTGAGAAACTT
CTGGAATTCTCAAGAACCTCTGAGAAATCTCCCATTCTGTGGCAATAAAG
TGGTGGTACTCAAGCCATCACCAACATGGCTCATATGTCTCATTGCACTAT
GTCCAAAGACAAATGGCCTCTGCTTCTCTTCTGTCTCCTAAATCTCTTAAG
ATTTCCTCTTATTTGTTTAAGTCTAACCCAGAAGACTTACATGGCAGGTAA
TTCTGGGAAATGTATTCCCTTGTTTCTCCAAAGGGGTGGGGATAATACCA
AGTTGACAACAGAAAATCTAGCACCCTTTTTAAGGTTTTGGCATCAGTGA
TTTCTTGGTGCTCTAAATAGTTCTTATGATTATTCCATGGGTGCTTAAAAA
GAATATGTATTCTCTGATTTAAGAATATGTAATTGATTTAACTATTGATTA
GATATTAATAAAATAGATTTTTAAATTACCTTATTAATTTGGTTAAGTCCT
CCATGACTACTTATTTTTTGACTATTTGTTATGGAAAGGTGTAGCTAAATA
AATATAGAACTACTCTATATGTGTTTCTAGTTCTCTATGTATTCTCTACATT
TTGCTTTATAATTGTTTTCTTTCTTAGAGACAGGGGTCTTGCTGTGTTGCCC
AGGCCAGAGTGCAGTGGCATGATCATAACTCACTGTAGCCTTAAACTCCT
GGGCTCAAGCAATCCTCCTGCCTCAGCTTCTCAAGTAGATGGGACTACAG
AAGTGCATCACCACACCCTGCTAATTTTTTTCTTATTTTTATTTTTAGTGAT
GGAGTCATGCTATGATGCTCAGAGTGGTCTTGAGCTCCTGGCCTCAAGCA
ATCCTCCCACCTCAGCCCCCTGAGTAGCTGGGATTATAGGTGAGAGGCAC
CATGCCTGGATTTGCTTTATAAATTTTGATATGTTACTTGGTGCATAGATA
TTCATTGCTGTTAGACCTTCATTATGTGTTGTACCCTGCCCTTCTTTGGTTC
ACTTTTCCTTGAATTCAGTCTTGTCTGATATTAATTTTCAGCTGTCTGCTTT
CTTCTTATTTCCATTTTTCTAATATGCTTTTGCCTACTCATTTGCTTTCAATT
TTTTCTGAGTCATTTCATTCTAGATGAGTATATTACAGTCAGTACTTAATT
GGGTTTTTCTTTGTCACTGAATATGAGTGTCTCTTTTCCTGTGTATTGATAG
GGTAGATATGTGTAGTATGAGTGTTACAATTTTTTAAAAAACTTTGATTTC
TCTCCTTTGTTTTATTTGGGGTCTCCGGTTATTTTTTAAGGTGTATAGTGTC
ATTTTATTTTTAATGTTTGCCTCTGTAAAAATAATTTTATAGGCTGTACTTA
ATCTTCTATTTATTTAGCTAGTTTATATTGACTCTTCAATAACAGTAGTAA
ATGTCCCTACTTCTAACTCCCCTTGCCCTCCACCTTTTAATTTTGTGGGGTA
TACTACTGGCCTTAGTTTTTCTAGTGGTTAATTTTATACATTTAAATAGGTT
TATACCTTTTAAACTTCAATGGCCACCTTTAAAAATATTTTGACCCCACCC
ACTATAAAAAATAAACATGCCATCTTACACGCACAATCTCACTTTCTCTCC
CCTTACATTCCCTAATTTTTACTGGGTATTTTAATATTTCTTCATCATCGTG
GTTGATTACATTTACATGTATATTGCAGTGGTAGTTCCCATAATTGTTATA
ACCTTTAACCATAGTTCTACATTTATTTTTGCCACCGTTTTTCCCATTCACC
TTTTGGTTCAACTTTATCTTCTAGTAGTGTCTTCAAAAAAGTGTTCTTGGA
AACTACAGCCCATGAAACATTATTGCATATTTGAAAATATTTATCTGTCAC
CATTACACCTGAATGACAATTTAGCTAGCTCAAATTTACTGGCTCACATCA
TCTTTTTTTTGGTAATTTTTTAAGTACTTCTCTACTGTCATGTAATGTTGTA
TGTAGGATAGCCTAAAGCCAACTTGTGTTTTTTTATTTTGCTGTTATAGTA
GTTTGATCATTTTTGCCTAGATGCCCCATAGGATTATTTTTCTTTGTCTTTA
TTTCAGGAAACCTTACTAGACTATGGTTCAGTGCTGACTAGACATTTTTTT
CCTTAAGAAACACCATGCCCTTTAGATCCTTAGATTCAAATCTTTTTTAAT
ATCAGTAAAGTTTTTAAATTTACATATTTGGATATTTTATACTTCTCTAGTT
TTTCTTCTTAAAGAAGAAAACATTAATTCTGTCTTTGCCTGTCTTCCATATC
TACAATTTTCTTCTAATCACTTTTTATAATTTCCATTTCATTTTGCTCAATT
GCCTCAAAGCTGCTATCTATTTTTCTGTTGTCAACAATCTGTACTCTTACAT
CAGCTACTATGATAATATTGTGTCACCATTTACTACTATTTACAAGCTAAC
CATAATTTCTCTGATGATTTTAGTTTTCTCTTCTACTTTGTGTCCTAGATAC
AGGCAGCTCAATTTTAATTTTTGTTGCTTTTCTTGATTCCTTATACCTCTGC
ATTAAGCTCTTAATTCACAAATTACTTCCATTGTTAAGTTATTGAGTTCTT
AAACAAATATTTGGTCATGATTTTCATCTACTCCACATCAAAATGTTCATA
CTGTGTAGGCTGTTTGCCTTTTTTGTCCTGCTATTTTCCACCATTTTGTGAC
ATTTTTGTATGGATCTCCTTGTTCGTTTTTTATTATTAGTCATCCTTGAGTG
AGCAGAAATCTTCCTAGAGCAGGTATTTGAAAGAAGTAGTGTGGAAGAG
GGGCTGGGGCTGTGTTCTGCGTTAGTAGAAAATCCCCTAATTCTTAGAAA
AATCCTTTATGATTCATGGTCTTGTGGGAGAAGGTTGTGAAGCGGGTTTTG
TGAATGTGCTTCATGTGCAATAGAGAGTGACTGAGAGGTGGTTTAACCTT
TCTTCTTAGCTCACAAAGAATGGCTGCTACAGAGCTGCTTAACACCCACA
GTTTTTCTTCTACATGCTGCACATTTACAAAGATGGAGTATCCGTGAGTTT
TCTCACTCATTCCTACCTCCCCTGCCATTTTAAAGTATCCAAGGAAGTTGG
GGAGATTCCCTCAGCTAAACAGCCACCCAGATCTTATTGTTCCAAACAAA
AAATTATTAGATTTGCCCTCTGAGCAGGTCACCTACTTTGGAGAATTTTGT
GCTCTGCCAGCTCTCCTCAAGATTTGGAAAAGCAGTTATTTTCTATTGGAG
CCCCCATCACTCCAGTCCAAAGTTCACTGCATGTGACATCCCATCTTATTT
GTCTGGTTCCTGATTATAGGAGTTTCCCAGTTTCAACAAAAATACTGTCTT
TGTTTTTGTTTCTCATTCATTTTTCCCCAATTTTGTTTCAAAGCGAAAAGTA
AAAACATTCTTACTCTAACATCTTAAAATTAATGTCTGGGAGGAACCTAA
ACTGCTCATTAATATATGCCATGCTATATGTGATATGGCAGAGACTGCTAC
CTGTCCACCAGAATCTATTTATTTTTCTTGGATGCACAATAAGACTACATT
TCCTAGTCTCCCTTGCAATTACATGTGACCATGTCATTAAGATTTAGTGGA
TGAAATTTAAGCAGAAGCAAGGTGTGTAAATTCTAGGGCTGGCCCATTAC
AAACTTTAACATGGGCACCTGTGTGTTTTTCCTTTTCTGCTTAATTGGGTTG
GAGATAATCACAGTGATGCTGAAAGCCATGTGCTAAAGAAGGCAGAGCC
TCCTTCTGCTTGGGCTCCTGAATGACTGTGTGGAACAGAGGCCCTTGCCAC
TCTTGGGCCATTCTGGATTATTACATGCACAAAGAGTAAAGTTCTTTTCTG
TTAAGCCATTAGATGTATGGGTTTATTTGCTGTTACAGCCTAGTCGAACCC
AAATGATGTAGTGAGGGAGGCAGGACAAGGAAGAAACCTATTCAGTATG
GAGTTATCCTTAGGGACAAGTTACAAAAATTATAAGGATCAGACACCACT
GCTGGTTGATCCATTATAGACCAGAATAAGCAGGTGGTATGATTCCAGAA
GGAGATGCCATGGTTGTGTGAGAGAGAGATTAACCATTCCTATAAACAGA
AGGGTCAAAGAAAGTAGCCAGTCACCAGAATGCTTATATTAAGGAGGATT
GTGACTACCTGTATATAAAGTATAGGAAGAAAATGGGGCAGGGCAATTA
AGATGAATTGCAGACACAACTATTTGTGCATATTAATTTGAATTTAAATTT
AAACTTAATTTAAGGATAATTTAAAGGAAAATCTTCCCTTACCTATTTCAT
GCTGAAGGGGTCTTTCAAGTTGTCAAACTCTCTCTATATAAATAAGACTGC
ATAATCCAGACCCTCCGGAATCCAGAGATGAATTGCATGTGGTAGCTGAT
GTGTGGGAAGCCCACAGAGCAGCACTTCTGGTCATTGCCATCCAGCTAGC
TGGTGCTCTCAGAAGAATGAGATGAAGAGGCATGACTTTCAAGCTTCCTC
TGCCATACAAGGTGAGTTTGGGTTCCAGGCTTCTTCCTAGCCCCTGTTTCT
GCTTCCTCTGATTTAATACCCAGTCACTGAGTACCTACTAAACACTCGGCA
TTAAACTGAAAAGAACAGGGAAGTCAAAAACCTTTTACCAATGCCTGATG
GGATAAAGACATGACCCATCCCTCAAAGAGCTTGTGGTCTTCCCATGGCA
ATGCCCTTCCTCCCTAGGCCAGATGGACTGTGAAGAACAGCAGTCTCAAG
TGGATTTATTGGGAAGCTCATGAAGACTCAGCTCAATGGGCCATGTTTGC
AGGGGCCCCTTACAAGGCTCTGCACTTGATTTTGTAATTTTGAATTATTTT
TCTCAAAAAGGCCTCCCAAATTATGTAAGCTTCAGGCCTTTAAAACTTGG
ATCTGTTCCCTTGAAGCCACATAATCTGATCAGAAATAATTTTCAGGGCTC
ATTGGGTGCTGGAGAATTCATGGTTAGATATTTAAATAAAAATGCAGAGA
ATCTATAGGACTTTCCAACTGAGACCAAGGCTTTTTTTTTCTGGTAGCTTG
ACTGCCAGGAAACAGAAAGCGGGGCATCTCCACCTGCTGGCAGAAGACC
AAAACTACACCATATTGCATTGACCGCAGACATCAGGCTTTGTTTCCTTTT
AAGTGAATAGGTGAAATCAATCAAGCAAACTGGAATTAGGAAACAGAAG
GACCACTCACAACCGTACCTCCACAGCAAAGCTAACACTTTCATTTTGAA
TAACCCCTTTCCAGCCCCTGTTTACATCAGTCACATTTTTTGCATGGACAT
AATAGTGTTTCAGGCAGTCCTGCTCTACAGAAGGCATTTGTTTCGCTGACC
ATAAGCATCTTATGTGCTTCTACATTGTCTTTATAGCCAGCTGTCTGAAGT
TCTACATCATCCGTCAGTGATGGGTCCTGAGTAAACTCAGCAGTCTCCTCT
AAGTAACTTGTATATATCCCTTTTATAAAGAATGCTGCAGTGGCCACCCTT
AAGTATAGAACTTTGTCTTTCCCAGGCATTACTTACAGGAAACTGGGGAC
CAAATGCCACTCCATAGCATAGCTTAGGAGAAGTGAGATTCCAAAGGACA
GAGTGGGCTGGCACATATGTAGTAGAAGACTGGACAAATGAGAGTGAAT
GAGCGAGTCTGTTGTGAGTGGATGACACGCAATGCAACCCCAAGGGCCG
GCTCCAATTTGCACAGTCCCTAAAACATCCCAGGGATCCAGTCTTATCGC
AGTCTCATCAACATTGACAATAATCATTTAGAGAATTTTCACCATGAGACT
TTTGACACTCTGCTCTGGGATTTTGGAGTTCCAACTGCTTTAACAATTTAT
ACAGTCACTCAAATGTAGAATCCAGTGAATTATTAATGACAATTTTGCTCC
GCATATTGAAAGGGCATTCTATATTAATCAATTATAAATCATGATAAATTG
CTTTCTAAAAATTATACACTTGAAACCCAGAAATGTTCAAGCCCCTCACC
AGTTTACATACATCCTTAACCGTGTCTTGTTAACTGGGTGACCAGGAAAC
AGAGAAAGGAGGATGTACTTAATGAATAACTGGCCAGAAGTCGGAAGAC
CTAGATTTTCTTCCCATCCCTTCCATAGATTTGCTCTGTGACCCTAGACAA
TTCATATGCCCCCTCTGTTACCATACTTTTAAGCTGAAGGGGTTATAGTGC
ATACTTTCTGAGGTCCTTCTAACACTGGAGATCTATGAGTTCATAAGATCA
CATAGAGTCTTGGACTTTTCAGAGATCACTAGGCATAAGAAAGCCCTCGA
GGACATCCTCTTCCTAGATCAACCACTCCAAGCTAAGACCTTAATGGGTCT
ACACTGTCATCCATTTATATAGGAGAGAGGATGGTACAAGCTAAGTGAAA
GTCTGTTGTCAACTCAGGAACTAGGGTACAGTTCCAGCAATCAATCTCCA
TGGAGGGTTAGCAGAGGTCTGGCCTAGCCAATTAGGGTTTGGCACTGCCA
TATGCTACTAGAAAACACATGCTTTCATGCCAGGCAACAGGGTGGGAAGT
GCAATGCCATTAAGGACCATACCTTAGCCTACTTAAACTAGCCTGGGATT
GAGTTACTGAGCTCAAAATACAATTCAGCACACTTTCCTGCCCACTGCAA
GTCAGGCCAGCCCCTGGAGAACTCCAGGGACAAGGCAAGTCTGGAGTTTG
TCCCCAAGGAACTCCCAGTCTAGTGGAGGAGATGGGCCCATAGCAGAGTT
ACAAATGTCCAAAATAAGAGCACAAATTCCTGGGTTCTTAAAGAGAGTTC
AAGCACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAT
TAATTTAGATAAGGAAGGGAGGTAGGGAGAGATGGTGCCAGCAATGGCT
TCCTGAGGAAATGTCACTTGTGATGGGTCTTTTAGGTGAGTTGGACATTGA
GAGAAAGTAATCAAAAGGGCACAAGATAGGGTAGCTGTCAGGTAAACAA
AACAAAACTGTATGCACAAAGGTCTAGAGGCAAGAAAGCATGGGTGTGC
TTGCATAGCAATGAAGAATGTGGTTTGGCCAAAGCATAGGATTCACAGAA
GGACCTCCCCAGGCCATCAGTAGTCTCTCCTTCGCAGGAGTCCACTGTGG
CTGGACGAGGAACAGATCAAAACAGGTCTTGTTTGAGGCTTGGCCCAACA
ACAGGGCCAGCTGTTGACCAGGGCTTCCTTCACCCTTAGCTTTCTCTCCCC
CTAGTGTAGTCAGAGACTCCAAAGGCAGTCCCAGAGTGTTGTGCCAAAAC
AAATGCACATCAGGCTATTGTCATCAGGAGCCCTTGTCTTGTTCTGTTTGT
TTTAAGAAGACATAAGTGAAATAACCAGGTTTCCGAAGAGAGGGCAGAA
TTGGGTTTGTCCCCAGGGAAAATGAGGTTTCTCCACCATGGCTGTTCTCCC
CATGCAGCAGCGGCCAGGGGTCCCCAGCAGCTCTTTGCTCAGTGCCCCTG
ATGCTAGGCCTGATGCTTCCTATGTGGTCAGCTCAGATGTGCAACCCTTCT
CCATTTTTATTATTTGAGCAATATAATCATTATTGCTTTTGTTACCCAAAAT
CTTATAATCACCCATTTAAGAGTAATGTAGGAATCATTTTTCTCTTTTATTT
GATGAACTAATCTTTCTGGTTGGCTCTTGAGGTGGGAGGGGTGGGCAATG
GGGACCAGCCTGCTTCATCTCACTCTTTCCTAGGCCTGCTCCTTAGGCACG
TGCCCCACAAATCCCACCTCCGCAGGTTGGAAAAGGGGCCCACAGAGGAT
CTCAGGTATCTGAGGAAGGGCCAGAGGCTATTTGGCCTGACATCCTGGAA
CATCACCAGCCAGGAGCCATAACTCCTTTAGGCTAGCCTGCCTGCAACTG
GATCAGGTATAGCAAAAACGATTGATCTCACCTTCTAATTTTCAATGAGT
GGGAGTGGCTGCCTTGAGTGCTGTGTTGGGAAGGAGTCTGAGGCAGCATC
TGGACCCTGTGTAAATATGCGTTTCCTGTTATTGATGAAGCTTTCAAGACA
AGATTTTAGAAAGCTTTGGCTCAAGTTGTAGGTTGTGGGAGGAAGGAATT
AGTAAGAGCCATCATAAACTCAACATCTATTGTGTTCCAGGAGCTGTGAC
CACTCCCTTCTTTTTAGTCGGGAAGGTCAGGATCTCACTATGTCACCTAGG
CTAGAGTGCAATGGTGTGATTATTGCTCATTGTAGTCTTGAATTTGAATTC
CTGGGCTCAAGTAATCCTCCTGCCTCAGCCTTCTGAGTAGGTGGGACTACT
GGTGTGTGCCACCACACTCGGCTTACTCCTCCCTTTGTATCCTGCTGCATC
GTTAGTGCATGATACCCACCACCCCCTCAGAGGCCCAGCCTAGTGCTGTC
TCTCAGAAAAACACCCTTTGAGTCAACAGAGACAAACTTAGGGGGTGCCT
GACTCCAAAGTCCAGGCCTTCCTCCTTTGAGGAACAGCATGGCTTCCCCA
GGGAATATCATTCCATGGAGACAAGGCCTGCAGACAGATCTCATTTTCAT
CTGTTCTTTTACTAGCCCTTGGAGGATTTGCTCCAAAAACCTGAGGCACTG
ATGTTTACTCCAGCTTTACTTCCCCCATACCTGGTGAACAACTCTGGCCCA
GCTTCCACACACCAAGGCCCATGGCTCTAATAGAGCCAGAGAGGCAGTCT
CAGCTAAGAAGAGCTGTATCCTCCAGGGACCAGCAGGAACTTCCAGAGAT
ATCCTACAAGCTCCAACTGCTGGGAAAAGCAGACTGGGGTCTTCCTCAGG
CTCCACCAAAGATAGCCAAGTGGCAGCAATGACATGAGCACCTCATTCCT
GTCCTAGACCCTGGCAATTCCATGAGAGGGAGGCTCTGAATTCACCTCTG
TTCCCAGGATGTGTGACATCAATGCACATTGTCATACCAGATACACCTGC
CAAACTTCAGAACCCCTGGGAAGTGACCAGAATGCCACTGAATTCAACAC
CTTAGACCAATGAACATGACCTGGGTCCTGTAGGATAGGAGGCAGCAGCA
GTGTATACCCAGACCTGGTCGACTCGAATAGATCTGCTAGGCGCCAAGCT
TGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGA
CACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGA
GCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAA
AAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAA
AAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTAA
CTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCC
CTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAA
ACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTA
TTGATGATGTTT
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.
SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> IMPROVED HELPER DEPENDENT VECTOR SYSTEM
FOR GENE THERAPY
<130> 20377 PCT
<150> 60/118,601 <151> 1999-02-04 <150> 60/138,134 <151> 1999-06-08 <160> 17 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> Consensus sequence <221> misc_feature <222> (1) . . (15) <223> n = A,T,C or G
<400> 1 atttgnnnnn nnngc 15 <210> 2 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Adenovirus 5 <400> 2 attttgtgtt l0 <210> 3 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Consensus sequence <400> 3 attttgttgt 10 <210> 4 <211> 158 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic packaging signal <400> 4 gtacacagga agtgactttt aacgcgcggt ttgttacgga tgttgtagta aatttgtcta 60 gggccgagta agatttgacc gtttacgcgg ggactttgaa taagagcgag tgaaatctga 120 ataattttgt tgtactcata gcgcgtaatc tctagacg 158 <210> 5 <211> 158 <212> DNA
<213> Artificial Sequence <220>
<223> Adenovirus 5 <400> 5 gtacacagga agtgacaatt ttcgcgcggt tttaggcgga tgttgtagta aatttgggcg 60 taaccgagta agatttggcc attttcgcgg gaaaactgaa taagaggaag tgaaatctga 120 ataattttgt gttactcata gcgcgtaatc tctagacg 158 <210> 6 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> Linker <400> 6 agctcggccg attattggcg cgccagatct gcggccgctt ctagaaacgc gtgaattcgg 60 cgcca 65 <210> 7 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> Linker <400> 7 agcttggcgc cgaattcacg cgtttctaga agcggccgca gatctggcgc gccaataatc 60 ggccg <210> 8 <211> 40 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 8 attggcgcgc cttctttctg ggatgattcagcatcaactc 40 <210> 9 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 9 gatcgtcggc cgcttgggtc atagacttctttgagaacca g 41 <210> 10 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 10 atcagttagc ggccgcacaa gctaagatcacaaagctgtt t 41 <210> 11 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 11 tatggcgcgc cgctgacacc cagcctgggtgccggtg 37 <210> 12 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 12 tcgacgcgta gcgctgtgtg gccttggcagtttccatag 39 <210> 13 <211> 45 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 13 tcagtaatgc ggccgcggga tcattcctgg actcagattg ttctg 45 <210> 14 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 14 tattaaggcg ccgggcatgg gagtgatctc accaactctg g 41 <210> 15 <211> 46 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 15 tcgacgcgta tttaaatgtg ctggagtgtt gagatactgt agtggt 46 <210> 16 <211> 28068 <212> DNA
<213> Artificial Sequence <220>
<223> Modified adenovirus <400>
aaacatcatcaataatataccttattttggattgaagccaatatgataatgagggggtgg 60 agtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaag 120 tgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgttt 180 ttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttg 240 tagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataaga 300 ggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgc 360 ggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcg 420 ttccgggtcaaagttggcgttttgattcggccgcttgggtcatagacttctttgagaacc 480 agttataagctatggtttctctccacagaaaaagcacttatggtgtctccccctttccag 540 cccaccaacattttacatctaatttgggggggttttctggaccacttaatacccatccat 600 ggatctcatgtgaagactcccctggcttgagaaatcactgtcttgttgaaaatgggaaca 660 aagctaagtcagatagctggttcatcagcaatgactttgaccaagcctgatcccacccta 720 ccccaccccaccccagtgaccaccccccacaatggagcacacaactctaaactggtttgt 780 aggtatgtgtgtgtgaacacgctgaggaatctgcaaaaccaaatggtgagtgcaaaacca 840 aacagtcacgagtaaatctcacaacaaccacgtcctgagctgcagcccttgttgaactat 900 accccactagggccccaagattttaggacttgtgtgtgggtgggacctcccctttctatc 960 atgctttagaagacagggatttcaccagaattgaacatattgaacatatgacccattttt 1020 ttcagccaaaggcaattaaaataacttcatacttgatatccatgtcagcaaaagctgcaa 1080 aacgcaaatgggtggctgctaagagccctggtaccctgacgagcacaccaagtgcttagc 1140 aacagtggtgtccaaaggaccagctggaagcctgccttgatgagaagttgctcttctttc 1200 tacatgaaggaacacctctactctcctgcttttaatacctgagctgtgagtgatcatcta 1260 tgtccattagcaaacatcccagtggagaaggaaacactcatacccgaaatctaagctaca 1320 tagttggaatcacttcaacttattgcaataaacacttactaagcacctattgtgggcaag 1380 tctttgcaatggataatagttcagtagatattttgatgtaatatttgaaataacaataaa 1440 aattgccaccactgaatttattgagcatttgctgtgctttaggcactaacccaggttctt 1500 taaatatttggtcttattcgatctgtataaatagccatctatgagaaagggactattatt 1560 gcccttattttacaaatgaggccaatgaggcccagagaggttaactaagttgcccaaaat 1620 catacagcccactagtggcagagcaggatgcaaacccaggcttgcctcgttcccaagccc 1680 acatgtcgtttgcattggtgtggaggtgtgcatgtgtttagtcattagcatgttatatga 1740 taagcaagttttgaaacatagaaacttaaaatgtgccattaagaaaagtacaggcaaggt 1800 tttccaaggggaggtgtggacctccggacaaatttttaagaactaattataaatacttaa 1860 aaatgggaataagaagacaacctaactacctgaacagttttagagatgactcatgcccac 1920 cctctaaaacccaaacaaaaacaacaaagtcaagaaaacccatgaaatcttagcaagcga 1980 tttctatgtacttgtgaaaaggatttctttaccattctaatgggatttatgccaaccata 2040 gagggctcagtgcccctcccatggggtggttagtgagtacagagctgagctcaccggcca 2100 tctgcagcttcatgttatcaagctccagtttgtccttggagcaaggttatctgggacatg 2160 agcagaggcattgctttctgcaatggacagttctttctgcctgcatacctagctccttga 2220 taactttaaataccattttatagccacactggagttttgaagacctcaatatgcaaatat 2280 tactcaggttctgattacttgtctgctccatgataacacactctaaaagcaatgaatggg 2340 gcttatttgtagagaactgaagcattttaagcttttgctcaggaatccctggtagcttcc 2400 tgtgacttgcaagatattagtgatgggtcaagaaacaggaccccccatcagcataacata 2460 cgcagtgcctcagtagtccatcaggcagaaaaaactgcagatggcacatggaaatgacca 2520 gcggcggaagataccccgacagtgtgggcagttctatttcagcagcaatcaagagggggc 2580 ctggagccactcaatcaagtggagcaggatgggagcaagcactgtgcagaccaatgcaat 2640 ccccagttaacacaaaaaataaataaaagagatgagattcagtctcttgactgtgactga 2700 ctgggagctttatagctgatgcttgtgtcttttctccattttatttaattaggaaaagaa 2760 atgcttatcacacactctacgtgtgaggtacaacctccacaggaaaggttgttaggaaca 2820 tttcaacttctagaagtttctaaacataaggtaaatccatctttgtccttgggatcactg 2880 cacatctcagaaaggcaaataaatcagtaattggtgggcataattactagctcatggact 2940 gacaaggtctacactatttcgaatctcacagaagtaagccatgggacagatagagtctga 3000 tagtggtgccccgtttcctggaggtcacacttactcatccccctggaccctgggcttctc 3060 atgattgtcagagagtttgctggaaccaggtcagcccagtttcccttcccctgaaaaatc 3120 ctccaatggctcgcaccaagactagagatgcaagtgcaagcacatccaccctctcagcag 3180 ccaggttttgcgttccataatgtcacgtacccccagtcataccaatctccttggagctct 3240 ccagacaggctgccatgtgtggtcggccctctgtgcttgtgctccttggtttgccaagcc 3300 tggaatgccctttctccatgattgtactctgggaattctgtgtgtctttcgagatgaagc 3360 tcctccaccctggaaattctgcctctctttccaggtccagctcctgtggctgaataccct 3420 tggctgactgaacattcttgctgggccaagtcttaatcatctctaaatccctctggtgcc 3480 ccgacagtatctagcccaggcaaagggctcagcaaatacttgcaaaattcaataaacttt 3540 actatgttactagcttggcatgatgttctaggcacttgggagattataatctggtgagca 3600 ttgtgctagtcttttttttgggataaacaccaatgataaactgagcctttactctcattt 3660 tagcacatctactcatctcttttcaatgctggtgggtttataaaacccatcagtagaaca 3720 gagagtgatcataatcatatggtgaaaataacatcagctaacatatttactgaacatgct 3780 ttagtgtgctgggcactgaactctgtgcacatgtgaatttgagatagattgttcctagct 3840 aataagatgaggaagtggagatgggcttactcaagtcacacaatagcaagatggggtaca 3900 gacagaaaccaagctagaaaccaagcaacccctgggtttggaaatgcatgggctcctcct 3960 ctgcacatggcgaggagcagtcaggtgctcctccttctctcatactgaaataactctgca 4020 cttttggctattctgtgacactctgtgctattctgtagctcaaatggctctggtgcaagg 4080 agctcagattatgactaacccccatagatattcagctgctttgcaagaagtggatgaatg 4140 ctctggctttataaattattgactagattggatattggcccaatctccactctggtgatt 4200 cggaaggaggcatatgcacatttgcaaggttatagtagtgcactctaattccactggctt 4260 ctgagagcttgtaggtttcttgacttaatcattctggaattaaggtaatggatcctcaac 4320 accttttctttccctggcctttactaaccatgtaacagaaatgagcagagaaaacccaag4380 aaagcgaactggagacttgatgagtgtgtcaaagatgctcagagtccaaggtcctctgtg4440 gctcactgacttcagaagccaacctccgttgttcaagtcacttgtgaggttactatgcta4500 gagcactaaatattatccagatagcccaaagaggtgaaggcagacatgtggaagaacctg4560 gatttttgggcaagttgatgaatgcctcctgccccatatcaaagagaggtgataggagac4620 aactttgtgaatttgaaataatgcaccggggaaacaggaaaacgtaatgtaagtcacgct4680 tcttggcttttttcttgccattaccctacttggccaagtgcaaatgggatttcaatatat4740 cataagtatgcatctattaataacaatgcaagaaagctgtacaactgaagtctgagattt4800 tgtaagaaacagaatctctgtaagcatcaccatccaacagaacttcctgagttgatgctg4860 aatcatcccagaaagaaggcgcgccagctctgcaggatcttcaagtctggggtgccacca4920 gcaagcgacggtcctccatgggctcttcaccttacggcagtgtccagaggcaccgccagt4980 cctctgctcctatgctggtcctgctgtccctggcaaaaggagccagagcattctctccag5040 gcctcccgaggaggctgcttcctttgttttgcagatggaggctcccatcctttgttctga5100 atcaatgtgctccaaagataagccccaagaaaacagttgttgccttttgacactgacaat5160 tagaatcgttggaaaatggagaaaacaggaaatggcaaatggtttcagtgaccaggagga5220 aaccgtgcctgaaagttgctgcttagtgactgggacactcgctttctgctctcttatgaa5280 ggacagcctaggccgtgtggccttttataaacaaagctatgaaggggtcgtcaaattttc5340 tagggctgcaactgtggcactacgtcctgttgtgccaggtgacactgacaagcagcactg5400 agttctatgcaagcccaggtgtgcttctctcatggtgacccccagagaactaaggcccag5460 ctcttcctctgtcacacccctcccagcccccactgtcagacaagggaccacattcacaga5520 cagtctcagccaagatggcaaccttggaagtcctggggatgcctttctagaagctttagg5580 ctgacgccagggagctgtaaagcccccacctgtccctgggggttgtgtgctgggcaggag5640 agaggggaaagagcccaaactccaccactgcctgctggcacaactgagcccacgccaggc5700 actgcaccagcttcactcaccagctaccacatgctaatgtctcctgtttacccaggtgca5760 atcacctgagtgctgctttctcaccactgcatgggaagaacgatctcattaccaattcaa5820 ccacttaagcaggcaaaaggactccaacgggggaaaggcagaggacaaacgattcgggga5880 ctgagacagatgcccacaaaatgttcctggattttagccggattcgggctgaagttcctt5940 gccctatgagcccttggaggagagcatcctgactcagacaagaattcaaatgacaagact6000 tctgggggatggcagtgaatggatccaaagccttgttttataaagaatgtggaggaaggg6060 aggaaggaagggagggaaggggagactgagagggagagagaataaagaagaggaagagaa6120 ggaagaggaggaggaggaaggggaggggagaggaaaaaggggagggtagaggaggaggga6180 gaaggggaagggaggacagaagaggggagggagcggggagattatctacccctccatgct6240 agggaactcctgtctccttttccagcagacactgctccatgagtgcccccacagagagcc6300 agcctgccacacagcagaaggtgctcagctatctgtcccaaaggttgagcactagtgtat6360 accacaaggaaaaaaaccttcttttgtactgtttggaagccagaattagaaacaactcgt6420 tcacccttctgactttccctttgcaatgacaggaagatagtatactgcatgggaccacca6480 cacattcaagatctgagttgtggggacccccacccccacatgggtggagccccctgagac6540 ccgtgggctctgactggccatggcccatagatagtgacaacatacacactgctatctgaa6600 gcctagttggaggacggatggatgcttttaggaaaaaattccctttagtcgtccctcaga6660 aactacaacggcttcctaaaatgaatggcaggcaagttttgattgtttcctaagttcccc6720 ctaactctcagagcctgtgactcagcttctccagaatgcctgcagggaggcaggaccact6780 gggagtaatgagcacgaattgggccacttctcccagtggactgttaacgccgagatctgg6840 ggtggaacccactgaacacctgcccttttgcgttgcccagaatgcagccagagggatacg6900 ggccacagtaactctggggcccctgaacctaccttcccaagaccctgacgggaaggtatt6960 gcttacatgacagtccagtcagtccctctgtcactcagttaacaaacatggtcatgggtc7020 tactatgggccaggctctgttccgggcatggggataacaccgtgtgtctctactcttatg7080 gacacaagtaaatcaatcaaagcagataatgttctaatgaccacccagtagggtgaaggg7140 atggagggtgggcaggtgctggcagaaacctaaggtcaagtcatgggaccccaatgggga7200 actggaaactgccacaccattttctatgttaggggtctttccttgcccttctagagacct7260 cacccatggtaatgatgctgccctcaggtttgaactggtctgaagctgtcactgggggct7320 tcacaaaatgaatgccactgaaagcaaacggcagagatctgtatatgaaattctttttac7380 cctttgttttctgtcttcccacatgatctatgactgctgcaaactctattctgactcctc7440 ctcagtcttggccggtctgggaagaacttggtttagatgctgggaatgatcccgggcaaa7500 ggaaacaaatatatttccagcttgcttttcttcttcccctgtggtgcctgcatcctctgt7560 ctcgcactgactcacaggctggactgacaaagtggctgtgttgacagggaggatttagtg7620 gatgccaggcctgaccctcaaaggccctcaggtggggccaatttccaaggttcttagaga7680 agatttggggccacaaatctgctctgccacttcactgctgggtgaaagctgttggcttga7740 gaagagagagaataggtgagggacaataattctcaaggcaagctagctgatatcctttca7800 tttgtggcttccaaagacagggaaaggcaaggcagaccagggtggaaacagcgacatcct7860 gaggggacgacgtccgttttgctaattctgtttccctggcagctttgtgctggacaagat7920 ttctctggtttcagctgaagagtgaacagctgaggagctctgtgactctgagagagagag7980 aaatcaatagcccttgaaaggaaagcactgcttgttcttccaggccagggcaggcaggat8040 aaagagaacaaggctgtacataccaaagccgaatttccattaaaggccagaatcctggga8100 ggccctttcagcttaatgaccatcttctgatcactatcacttagaaaatgttcatgtctc8160 cagttttctctgcagatagtgcagacatcctgtgcacattgatattttatgcaccagatg8220 caattctctgcaaaggtcccctctggctggtgtccatccgcttgctctcatgaccactga8280 tgcgactctgggggcaagtcaagccattccttctagactctagaatctagaggatgatcc8340 caactcacaggcctcttggacaagcttgctttggattccttctgcctgtactacccagac8400 tgccagtcctccagcagggcaatccaacttcctgcctctagggtgtgggagtcctgggcg8460 gggtttggaatccacagcagatggctggcaggagaggcagttcccgagcacccactaggt8520 gccaagcaggcaggtatcaggtgcagggcttcggaggtgcagaagcacagcccctgtccc8580 cctcaggttcccagtctggcctcagggacagacaggcaggtaggtatggaaacggggtac8640 tggaaggcatacagctgagggagagagcagctgcaccctggtcaatcccccactcatttc8700 cctggaggtggggctgaggcacctggagcactctgcttgcttgggttgtccctttgccaa8760 agaatgccgggcagaaagtagagaacaagcaggcccaggaagcactagaagtccctcatg8820 ggtggacagagctttaccttcggagctgtcacctcctgtgttcctcccaacaaccctgtg8880 aggcaggcgggcaggctgagcaggggactgtgccccgttgacagatgaggaaagcgaggc8940 tcagggggaagtgacttacctgtggcgtcgcagctaggcagtagtggagcgggcacctgc9000 ctccaggccttcggatcccagctgccccagggcgctgcctctgaggtgctgtacgaggag9060 gtgactcgggccagctcatgagcaaaggagctgccgggcaggaaggactcactccccagg9120 gcctggcaggacgggggctgtcgcccgaggctgacctgggggtgcctggcttccccaagc9180 ttgcaggctttgtctacatgagtctacaacaaccgattgaacaatccatcagctgcttgg9240 ccacaaaatggttctgactgatcttgtcactgccctgagtgtctagctgggtcatctggg9300 tgttgtggttatacccaccaactgctttggtggaacgaaaccgcggccgcgggatcattc9360 ctggactcagattgttctgaagaagcccagttctgggtggcatcaagtgcttgctagatg9420 gggggcttgccttgatccggctacacttggaggtgacttgttcttggacggctacataca9480 gaaagagagaagtggggatgagttccaaaggcatcctcgacttcggctgtggccaccgga9540 gggtagctcctggcccaacacggacttctcacctcccgcccttggctctctactgagctc9600 ccccctgctccccaattcctcgccattcccctcatttctctgccctcagcctggactgca9660 gttcttctgggaagctgccccaactccctaggtctgtgctcaccaagagcagatcacact9720 ggactgaaatgccagctgatttgtctcttcaagaaaattggaagctcctggaggtcaggg9780 tccatgtctgcttttacactcagtgctctgtatgcaggcctggcactgcccaccctttga9840 caggtggtgcatattttgtagaaggaaggaaggggccaggtggggtgggctgggctggtg9900 gcgggagctagctcagcctcttagattctctacccgatggatgtgacctgggacagcaag9960 tgagtgtggtgagtgagtgcagacggtgctttgttcccctcttgtctcatagcctagatg10020 gcctctgagcccagatctggggctcagacaacatttgttcaactgaacggtaatgggttt10080 cctttctgaaggctgaaatctgggagctgacattctggactccctgagttctgaagagcc10140 tggggatggagagacacggagcagaagatggaaggtagagtcccaggtgcctaagatggg10200 gaatacatctcccctcattgtcatgagagtccactctagctgatatctactgtggccaat10260 atctaccggtacttttttggggtggacactgagtcatgcagcagtcttatggtttaccca10320 _ aggtcaggtaggggagacagtgcagtcagagcacaagcccagtgtgtctgacccacccaa10380 gaatccatgctcgtatctacaaaaatgattttttctcttgtaatggtgcctaggttcttt10440 tattatcatggcatgtgtatgtttttcaactaggttacaatctggccttataaggttaac10500 ctcctggaggccaccagccttcctgaaacttgtctgtgctgtccctgcaactggagtgtg10560 cctgatgtggcactccagcctggacaagtgggacacagactccgctgttatcaggcccaa10620 agatgtcttccataagaccagaagagcaatggtgtagaggtgtcatgggctacaataaag10680 atgctgacctcctgtctgagggcaagcagcctcttctggccctcagacaaatgctgagtg10740 ttcccaagactaccctcggcctggtccaatctcatcccactggtgcgtaagggttgctga10800 actcatgacttcttggctagcctgcaacctccacggagtgggaactacatcaggcatttt10860 gctaactgctgtatcctaggccaataaatgttgatcacatttatagctgccatggtaggg10920 _7_ tggggacccctgctatctatctgtggaggctctgggagcccctgacacaaactttctgaa10980 gcagagcctccccaaccccttttccattccctatacctgacagatggcccaggaacccat11040 tagaaatggaaggtcactgcagcagtatgtgaatgtgcgtgtgggagaagggcaggatca11100 gagccctgggggtgtggcagcccccaagtgattctaatccagatcctagggttgtttccc11160 tgtcccattgaaatagctgctttaaggggcctgactcagggaaatcagtctcttgaatta11220 agtggtgattttggagtcatttagaccaggccttcaattgggatcctgctcttagagttg11280 gatgaattatttaactgattttcagatctcctctttctcaatgctttcagaagcacagta11340 actgcttactctgaaatgaattctcaccccacttccacatatgcaccccttgcccacccc11400 tttgggaacactggccttaactgcttaccttcaaatggactcatctgttgggagatatat11460 gcattctgccgttcaggggtcattgccataagacctgatctctgttcctcttgctaaaca11520 gaagatgaaaaagacaaattagattacagctaccaattaataattagccttaggatcgct11580 gcgtggggacctaggacttggctttggtgcagcagaaagcatgaataaacacaccagcat11640 acactcgcatgcatgccccaccctctcgagcaaaattccacaggtataaataaagtaaga11700 ttctgcacctgggttaaaaacacaactgcaacagcatagaatggggcaggagagacagaa11760 cttaatagcaagagcacacagaaaaaagttttaggcattttggatgtccatctgctcagg11820 atgggtcagcagtgagatgcggtcaccaaaagaacaaatgtaacattaggctgcattaat11880 agaagcagagtatgtagaaggagggaggtgacagtcctatgctaactctgccttggccag11940 actatacccacaggagtctgggcatgccagtctcagggagacccagacagactggctgca12000 ttcagaggatggtaagtaatgagagtggggattggacttcaaactacccagacaaagaat12060 ggctgagcaagccaaggatgctgtggctggggcagagcagactgtgggctatgtagtggt12120 ggatacctagcctctgcagggctgtcatagggaaaggacattgagaagaggactgaggct12180 tgttcctggtggtcctggcatgaacggccagatgatcacatggtcaggtggacacagtct12240 ccaacactgggagtagccaaacacttactgccaacctcccgcccttctcctgactagttg12300 cagcataggcaattgggaggagcttcctgtctccatctgaaagctggctgggtgggcagg12360 gggaggagcgagccaagtttcaaggccgcagtttcagcactcagtctgggatcggctcaa12420 ggagcaaaggggaagaacatagccaggagggaataacatgaaggcccccagacccagaaa12480 aggcatgacttgctctgagaccctcagccggttggtgtcaggttgtgactcggatccagg12540 tctgactcccagtccagtgcttgaagcctcaccccacacagtgaggggagcccggccatc12600 tctgctcaactgctgccatctctctccccttctcaaccaccaaggcagctctgtctggga12660 gcacaagctccaagtccactttctggtctgtgtcccccccaagatgccagaggacttgcc12720 tctacaacacgggctgcccgtgcagtgcctgcttttccagcaaagggcttctgggaaccc12780 ttctctgcactcagtggggctggtgggagtggggcggggtagcgacccagtgcttgggac12840 tgtgcccagctctcaggcctggcagcagttcctggccttggttcctgccaaggcagagag12900 gacaaacacatggcaccgggaagactacaccagaagcgattccaccagactggggtttgc12960 ttttctatcccgcccttagcctgcttcctgtcctggtccctgcctccccctccactggag13020 ctgccgtgtgggcagtgaggggctgtttctcagctgccctatggagctgccctctccctg13080 ccaaagcattggcaaggcggcaaggggtgggggtggggatggggggtgggatctgccttc13140 tcaagctctcattatactgagcacgtctcacccattattttatgtcatctagcaacaccc13200 catgtggacactgaggagcatgggggtcacatgaccactgcccaaggccacaccatccgg13260 atctgcctgagatggtcagggttggcagccatttctgaaggcagtcctttcgctttggct13320 cttcttgtaccagtctcaggacatcagggcagaagatctacagtccccagcttactgatg13380 tgacagcagaggctcagagaggttaaatgacttgcccaaggtgacacggctaagaagtac13440 agtatctcctaactgcagaccaggtgcttctgctgcttctggggacagattcctgcgtgg13500 ctggctaggtctaaacggtccttaactccatccccaccggttgctgcattagtttcatca13560 aataacacagttgtacagaggtaggggttcaggggcaggggcagatggaggctggagagt13620 gtgactaaggaaacagcaggggaagtgcggtaaagtccgaagggagggacggaaagagaa13680 agccaagcccaggggcgtgccagacaaaaggaaaggccacgccggggcagggcaggcttc13740 agcgggtgctggggcgtcttcatcccgggaagcacacattccagaggaccccggagtcta13800 atggaaaagctggccagcctatcactatggaaactgccaaggccacacagcgctacgcgt13860 atttaaatgtgctggagtgttgagatactgtagtggtgggatgcttaagtgctggggtgc13920 tgggtgttgggatgcctaggtgctggggtgccgagatgctggagtactagtgtgctggga13980 tgctgaagtgctggggtcctgagatgctgcagtgctagggtgctgagattctgggctgct14040 ggagtgttggggtgctgggatgctggagtgctgagatgcttggacaatggggtgctggaa14100 tactatggtgctggggtgctggggtattgagatgctagggtactgggatgctgaagtgct14160 gagatcctggagtgctgggctgctgggccacaggctcttgaatccattcgtctgcccagg14220 _g_ ggaagaaaccagaagataaagagctaatgaaggagctttggttgagagggaggaagtaat14280 ggaaggagcaacatcttgtggaggagcaggagagaatggacctcaggttgggagagaggg14340 ccaggctacaggccagagaggcagaaggattccagcagagtgtgggctccaggagccaag14400 gggaaacaggtttctgggaggagagagtccagtactgctgaagtggcaagtccgctgagg14460 accaggaagcttcatttggctttatgaccaggaggaatttggaactgtgactagagtact14520 tagggggaaggaggcaagactggagccagattgctctgggttgaggggtgagtgggaggt14580 gaagcagggcactgtcactcctttgaagggtggcagagagctggaattggtgctggatgg14640 gctgtggggtgacagggtcatgtggaaagcccctggggggcacctggaaaaggagaagct14700 gacagtacagtgagaggacagctaagggaaagcggaatggcagaacacgcactgccagga14760 ggaatgaggatagggtcaggagtgccaggggcagtgaggccagcctggggtcaggtggca14820 ggacgtgtccaggaagctggtctgcactgcagcccacactggctcagccttgaggttccc14880 tgtgtggttggggtaggaagttgaaccctctgggaatggaagatggaaccagctctgcga14940 gccaagctcagcttttatctatgggtctctgagggctggcagagctgagtggggacaact15000 gtgatccgtgaggctctcaggttgaggtggcccctccgggagggcttcattttcccagcg15060 ggtaggttctaagcagcagtggctgggcaggtgggtccaacacagagccagagaagggtg15120 aatgggcctcctggcaccccacccctgctgcccctgagctcagtgatggagggggacagc15180 acagctgagcccaagtgctttggtgtggccctgagggaaagctgcagcctgcctggggcc15240 tggcatggatgggacacttgaggcagagggacaatagtgggcgctgcagtgaggctggct15300 cttggagaggtttcctgaggagtgctgcctgagacgggcagggagaacagagacaaagtt15360 ggtgacagggaatgaaagctgactgaaggactttacccagacctatgaggatatctctct15420 cagcaggaagcaggaggggactgtgtgaggactggccaagagctggagtgttgggaaaat15480 gactctttctccgacccctctgtcctagctctggcccctggactgcggaggtctgcttcc15540 acccccattggtcgatcgttgtcccttgtcacagccattgagaattttggcagggagcat15600 gttcttagagcatttttaggctctgcgggacataacagctctgcctcagagcacatgcct15660 ttctcagctcctgaaagccactgatcaaattggaacattttgtaccttagggatgaggat15720 atcaactctcccagccacttagagggataaatgtgatgatgcattcaattgtgactacat15780 ctgatcccaactgttgcttcagctgctctcctatagcacatggcgggaggcgtgcatccc15840 agtagctacctccccacttttggggagatgtggttccatccatgaaacctgggtacccgc15900 ctaccaggtcctggcctatcaggtggcagggtctggtcaaagaagggcatgtgtggtctt15960 cagcaagggagacaggacggtggtgcagagcgtctagaccctcagggcaagtctccccca16020 cacctgctcccggggcagttgtctttgtgacctcccatccccctctgtttcatcctctat16080 aaaatgaggggctgagccccaaaataacaggcttctttgccatgatgcaaaactgctgaa16140 tctttctttctgacacacaaggcatcgagcagcctctgaaagaaccaaagccactagcag16200 gcttcctgacttgggtttgtaggtactgaatactcccttgaaaaataaaaacatagaggc16260 acttttctcctggctgtttattacagaacgaagaaaaaacacactggcttgaaacagacg16320 ccagatttcaaatgtagaggtgaaatacgaggtggcaattaaaatgtgattacagaaagt16380 ctggacactgagaaaagtttacaggacagtgggtgtgggttttctataacagacacttaa16440 atatacatgacgataattgcagatagaaaccatcaaagacaaaccccaaatcaactaata16500 atgtttacagatgttcccccccaaaccacagagccttacatcaaaacaaatactgaaagg16560 ctttaaaccaggaacagctcgccttaaccccacgagggtgcacacaagctgggctttttc16620 tctcggtctgaatggtaaagggaggaggatactctagctcctccaggtggattgctgaga16680 cagggctcggctcacacactgtctctgcgcctctcccaaatctggagaactctcccagcc16740 tcctggtaaagtgtctctgtggggcacttaacgataaaacagcttctgctgtaaagctca16800 ttaggaaagagctagcggagactgaaaggttcgcaaaagagattaagaatcacacaaggc16860 aataggatttttagtgaacatagaaataaatggccaagtggttttctatttggcatttgt16920 caacttgcacaacaactcttggtcatatccacattgctcattgcattaaaaccataagcg16980 actcagccacctagcttaacaaggtatcactggagcaaacaacacggtctgcatatttgt17040 aacattgtataataaacacaaaacaatgcatagtaaacacaactctactgaaacaaaagc17100 cgtcgctttatttacaaagtcacaaaatgaagtataaatacttctgtcattaatgtttag17160 gaaaaccatttacaaaattttcaaatatgtacacgtagcttgaaaaatcaccagctttcc17220 attttgtcacaggtagagagagggataagcatgggctgacaacaccactcaaattgtaac17280 gggagacaactgcgggtatggatcgacaccacttcctagagtgatgtcaccatgggggtt17340 tctatgggcatcctgctcagatttaaagtgccccagcatcctgggtgacttgcccagaat17400 tctgggctgtggcattttgagcagcagcatgctgttccaaaatgtcgtcgatcagcctca17460 agttgcacacccagtcttcatctgggctcacacaggagcctttcaagagagcttcaatga17520 aatctacctcattgcagtcaggtgacgaaatcagatcatttagtgggggttggggctggc17580 gcaaaaagtcggcaggtggcagctcagggggaatatccgttctgtcgaacggacctggga17640 actggctggcagcaacggcagaagcagcagcagcggtggcagcagcagccacatagcttg17700 gtggctcgatgccctgtatggggctcaggggactaaagctggccataccctgctggagga17760 acttggtggtgtttgctacaggcaccgggccctgtaccgggctctgcctgaggctctggc17820 tgcccagcaggctgaagctggggttgttggccaggggcacttgtgttcccatcgcagcgg17880 gcacttgtgcctcccaatcagatggcctctgaaggcaggcctggccagaaggtgagtgct17940 gctgaacgctattatccacttggctgaggggtgttttccccgaaactgctgtggtcacag18000 ctgctgccgctgtgacccatgcagcattgttgaacgcagtgggcattcttggcacactag18060 gccgtctgagctggtggggactcaaggactgggtgcccagggagctgggacagaacccag18120 gcaggggcacttctggtggggtggccttggggctctgcatatgctggcagacagagtcaa18180 gtctgcccaggggagtctggcctgagtgtgagaggatgggacactgggggctggaggtga18240 aaattccttgccgcttccccagagttggtgagatcactcccatgcccggcgcgcgcgtcg18300 acacgacagggacatactgggaatgcgcccttgccgtggaggcggggacccggcagcgct18360 acgtatccagcatcaacctgtatccagcatcaacccgccaagttcactaacttggtaggg18420 gtgaggttagggatccttaggagcccaggcagccagactttctggggagcccattcccat18480 ttgtgttgccaaagtacccccagcaggttgtgggaatgttgcctgtgaagagagtctgtt18540 ggggtgagatcttgtgtgtgtgcacagggtgacagttgtgtcccatttcccgggaagctg18600 tgatggcagcagaacctagaggagcctgagagagtgtgggagagtgggcctctggaagag18660 tagaggctgcggagccaggtgcagggctgtctgtcacccaaaggaagagggactgatgac18720 tcactgagcgtgtgtgtcccctggtggcagcaggccccatagtgaacataccataccttt18780 tctgtcctgagcgatgctcccagcagtcctgggagatggaacggtccttattcggctcac18840 aggaaggaccgccttaactggacagacacagcaaggtgctaaagatgccttccatcagag18900 gccaggttggaagctctaaagagacttctcttgctgttctctcacccacccccaggttgt18960 gtgtgtcccgctgtggattctcatgtcctttctgtgcctggtggtcctctactacattgt19020 gtggtccgtcttgttcttgcgctctatggatgtgattgcggacagcgcaggacacacata19080 accatggccctgagctggatgaccatcgtcgtgccccttcttacatttgaggtaagcgtt19140 ccacgggaagcctcttcagcccctgaagcttgcgcttcccctgacaggattctgcacccc19200 tagaaaggcagcctctgtccctcgagctcacagtgagcccactccaggagaggggagaga19260 acacagccatctccgagagggagcttcggtgaaaggagagcatccttcctttctcttggg19320 ggcagcacgtggggctggcagggagaagagtgcacctttttagccatggtgcctctgtat19380 ggctccagtttccactctggggaaagcagagtgggatgtcagatttgtgtattggagtca19440 cgtggagaattctagaatgggagctgttgactccttagaacaaacacccggaggagtttg19500 ccataaaactgctggcactgggaacttttcaagtggataggctattgccgagctctgaag19560 agggacataaaagctcatttcgagctttccccagggataggtggtttcctgcctttttct19620 ggcggtgctgatgttccctcttgtgggagctcacgcgggggtggggtggtggggaggaac19680 tgcctaatgaagtctggcttccgcctctgcccattttcggtgctggcatcaaccgggact19740 atgtctctttctttagattctgctggttcacaaactggatggccacaacgccttctcctg19800 catcccgatctttgtccccctttggctctcgttgatcacgctgatggcaaccacatttgg19860 acagaagggaggaaaccactgtatgtactcagcatttcagaagtccttggtgtgtgtctg19920 gggggggaccagggggtggggggtggcggatagaagtctaggaagggatgagtccccgag19980 ggccccaatttagaagcttgtgtgggaaagtgagggctgaggaaattctgggaccttcta20040 agggaagggcatgccgtaactctggtgttctgctggcctgcaccgggacttttctcgcag20100 tgcacgctgccatttgaggtagaaccagacacggcaggcaacctctcagagatcccgttc20160 cctcctctgcaaaatggggatcaagacagattcttcccaggcccgggagggtttgatgga20220 aaatccacatctcccacccaaacctgggattcatcctaggtccctgttggccgctctgcc20280 tcccccatatccttgctgccatcacccgagtcttgcctgtcttgccttgctaacactcta20340 ttcccctccacctgcttgctgaggcagacacttccaaaacgatctctgcagagggtgcct20400 tcctggcaaggctgtgggctccatggcacggaagcccagagcattgcccttcggaaagcc20460 agtgggtttgggggcagggcctcactgcagcccagcagcccgggctgtgcttgctgtttg20520 tgcctctgccccctaccccgcacccgggagcagggagggcttgcaccgagctgacactcc20580 agtagcctacagagaggagtagtgggactgggaaagtggctttaaggtggctccatgagt20640 tcaggccccctcctggccaacccgtgcatgactaccgccctcacggattccagagggtga20700 cagaaatcttgttcttgggtggcactgtcatccatgagtttatcctggctggagaagatt20760 agcggaagacaccgtagtctgcgcaccacagatattttgagactcactggagcagtagtt20820 ctcaaatttgggcatccagcagaatcccaaaagggccaggaaaaggggaccgctggagcc20880 caccctagcccgactcagtttctggaggtctgggctggggcccgagaatggcatccctaa20940 ctaggccccgtggacgctgtccctgccggtccgggaaccccactccaagcaccacagagc21000 tagcatttgcacttcttccccattttgggtactcaagccctgttcaggctttgtgactca21060 ggagtctggataaagtatgttatgacattgtaggagtgaaacttcttgttacggaaagaa21120 agttaacaggaaggtcagttgagcctcgtgtgtgaaataaaaaattcttatttttcaggg21180 tggtttggtatccgcaaagatttctgtcagtttctgcttgaaatcttcccatttctacga21240 gaatatggaaacatttcctatgatctccatcacgaagataatgaa~aaaccgaagagacc21300 ccagttccggagccccctaaaatcgcacccatgtttcgaaagaaggccagggtggtcatt21360 acccagagccctgggaagtatgtgctcccacctcccaaattaaatatcgaaatgccagat21420 tagatgccacttccggggacagagcttaagtggactgggacgcactctctccgccttcct21480 ctgccccctcgttcaccccgcagaccagaaccagtactggagctgggtctccaggtacgt21540 ccatctcatgccttgtttgcatccagcgcctatcagccactcaccacgacgggacgcgga21600 agtggcaggtgacgggggtgtgtgccagcagatgcggatgccaggaagagtgtgagaaca21660 ggggtgggattaccgtctgtctgggaggggctccaggtacccctcttccccgtcagaccc21720 actgggagatggctgcttgccaggcccccagaaggaacatctgtctatacggtgctgaaa21780 tcccaatcaaaagtattgtttagaaatgtatttctccacagggctgacctcctgcagctc21840 gctgagcactcccaggtcctcagcactcccaggtcgtggctggggcagtcagtaggaact21900 gtaactatgtctctgatgcaccacgtgtttagacacagcacagtccttttttctgttcct21960 actgtggaagtagtttctctttgggcatgctgacagcagtttttcatagcctcacggatg22020 agccctttctacgggagtgactccatgcttgtatacagagtatttatacaaatgttttag22080 catcttcatatgcggtgttaacccctagttctgtacagcatattctgttcaagtattttt22140 ttacaagcttgtgctgtaggcacatgccttctgctgcagaagtggacgcccgtggcacac22200 tccccccccccccccgtggggtgccacgccttcatgggacattgccacttctgccctgga22260 actcgtgcaggtacgtagtagctgctactgccacaacggcaacaccaagcaagagatggt22320 ccatgcttttctgacgttctcagaatagtggctagcttcaaacctgacaagcgctgcttg22380 aagccggaacactagagaatgttgctgagagcagaaacggccacgcgggtcacgactatg22440 cgtgggaaagtctcaagcttccctcctgccagcaacaagaaggctttggagtaggcatga22500 tgttttcacgtgtgcgtgccgtttctccaagcactgcaggttccaccgtgtgtcagaggc22560 tgcaagtttaacatcctcctgcctgaaaacaaataggtcctttgctgaaaagagggtaaa22620 aaaagagctttgatcttctcagccaggagaagagggtggtgttttcacgcgggcaactgc22680 tcgccggcctacatggggttaattcaagtctgctgcgagcacgactccgcccttggcact22740 ggcctccagcaagccctgttctctttggggtacaggggaacgggatggtttagactttcc22800 tgctcagtgtgtaaaaaatgtagctaaagccactatttttgctctccttaagctgttcaa22860 taaaccggttcctcattttacacgtgcatgatgtgtatcttctttgctggatgggccagg22920 aaactggagtggtcctctcagccagcctcagaggaaagaaatctctagctggcacaggca22980 gccagtgagtgaggctggcggctgcaggggcacagcctttagaatgagtccttcagtgca23040 caggtcccagggtatacggggtagtgggaggaaggaggggacgcctcgcagatgccactg23100 ttggctgggctacaccttgccacacttgttactgcttaggaggctttctggagtgttcct23160 tgggtgctacgacaatctgcagcagacactgtcctttcaccgctcctggtcctcgtttgc23220 tccccagtgatgtcaacagctgaggactgctcacgctgcaacaaaaggctctgcagtcgc23280 tgtctagcttgccctagtcgtctctagagttctgcctgaactgaaactcaagtggggttc23340 agctcatgacttgtggcaattgaccaggaaattcaccagttgctgtggctggaaggattt23400 tcagtcctgtgggttgtaaccagaggccacaggtggattctgccttaggctcatgagatt23460 tccgacttgctgttgaagaaaatgccttgtgaagtgacaacagtagctctgacccaactg23520 ccggtgcctcgctagttcctatacgtcccactggatcctcacagccccgggaagcaggtg23580 ctactactcttatccccgggaggagacagaggccgagagaggttaagtgacgtgcccaag23640 tcacacagctcggcagcggccgggttgagcatcagcagtctgtttgcagacccctcactg23700 tcaccccctgagccagtgcgccttgggccctgcggtcaggatgtctcaagcgtggaggca23760 tcaccggttcgtggcagtctctggaaggtcactgagctctgtgcccagaatcgagtcggg23820 ggagtctgtgcagaggtggccctgtgtgtggggacagtgtgtgacacagacactgctttg23880 gatggacacctctcccgtgacctcctagcatccaatcccaaaggaacaactgttgcagag23940 atggaccgctggacacaaacccacgtgcgtttctctggagacactggccaaggaaaacaa24000 aacatgctcgaaggccaacagctgcatgccccaccgcgatgtgaccgcagacacccgggg24060 tgtagaagggtctctgcctggtggggggacacgtgcaggccgaggagaggcaggaaggag24120 WO 00/46360 PCT/iJS00/02405 gctgcctccgactccccactggactgcatggcgacggcgtgtggtggggcagtcagctaa24180 gccatttgcctaaggggctgtcgggcatctgcgtgctggggaccgacagtgtgggtgtgt24240 taggaggatctgtatggagcacattgctgcctctggctaggacagggtggaaagggtggc24300 gtggctacagcctgacccatgggcaccgtcctaccctttgttctgtgcttccgagtgtca24360 gtcatgtgctggggtctgtgggcccatgactcagacggtgagctctgaccttcctgagcc24420 agggctttgctgtagttgtgcctggctcaggagctctaggacaaggggaccgctccaggt24480 ctgcatctacggtgtggcagggcccctcggcactcttgtgcactagtgtcatctttccca24540 ttgaaatgactgtgaggaccagaatgtgcacatgcagatgggcagctacttgtctgcctt24600 ggccctttattacacaacttgctgggggtggagatgccaccccccggcagtcagagcccc24660 tttatgatgtcatggggctggttacatgactgccaaggggtgctgctggccacactgcac24720 tagcaagtttgccagatggaggacaagcgatcattgagtatggctcgctgtgaagaaaga24780 aattcgagaggacaggatcatggcttggaaagggtgcctttccctccccagttgcagtca24840 gagacctaccttcacccagcagatccttcccctgcctgggacgacccggggtccactggg24900 agccctaacttgaggctgctgacagaagaaatcgctttccaacctctggccgaggaagct24960 tcgttcagaaggccgcaccctgacggtgacgtcccgccccagggagaagataatctcctc25020 tccctcccctttccacagaaactgtggagactggtcagcagcaaccagttttcgtccatc25080 tggtgggatgacagtggggcttgtagagtgatcaatcaaaaactctttgaaaaggagatt25140 ctcaaaagggacgtcgcacacaaagtgtttgccacaacttcgataaagagcttcttccgc25200 cagctaaacttgtatggcttccgaaaacggcgtcaatgcactttcaggaccttcacccgc25260 attttctccgcaaaaaggctggtctccatcttgaataaggtaatgaacgacaagcctctg25320 gaggggttaagtcggtgggctctggggcctggtcgggtggaagtcccaggactgcctcct25380 gggaagtgggcgacctcaggcagggtgtggggccatcgctgtgggcctgtgtccccctct25440 gggtggaggtgacatgaactaagagtgaatgtggggagagggctgaggatggtgcgggcc25500 cctctcgagtgtgtaaaatatcacaggtgccaagtagccgtatctgcgtgtcgtcctccc25560 cggggccagccatgtcatctggtggttgctgtgtccccctgactccacagcacattaccc25620 tgtgaggtgagcaggccaggggagtctggtatttgtaccactgtcaccctagctggtgtc25680 tggagaggtgctcaagtggaagcactgaagggcgcctggcgcaggaggtgcagatgctcc25740 tgctgcccttggtaggtgggcccctggtgtggaagagccagtacccagggcctccaaccc25800 agccggggtgcattctgttgccagctgacactgcatgggggaggcccagaatcttcttcc25860 ctcctggtctgcaacttcaaagaccctttccgccggccatggacaccctaatctgccatt25920 ttgaggctttttccaagacggaaaggcccgccacaacttggtaaaccttgacgatgtgaa25980 cgcgagtccccagcttcctttggggactgggaccttttccagaaaggcctcctgggccag26040 tagagttctcttgcacaggggcgtagatggttggtagttgtagtccatccttgtgacttg26100 cagtttccttttgtttatttaaactttgacttatagttagagttctactgccatccttac26160 tttcaaagagactcccctcacctcctcgtgaggatgaagagaagagtgggtgtcaagtct26220 gcaccaagacatcaggaggaggacaagccagaagctgctggatcctgtctggcaccagca26280 gacactgagcaacaagatcacacgtctccgaatgagaatgaccaggtcacaccgcaacac26340 cgggaaccggccggtcccaacacccaaatcaggagtggctctgctccaccagcaactcct26400 gtgatggtgcctgattccgccgtggcgagtgacaacagtccagtgacccagccggccggc26460 gagtggtcagagggcagccaggctcacgtcactccggtggccgctgtccctgggcctgca26520 gcgctgcccttcctctatgtccctggatctcccactcagatgaattcttacgggcctgtg26580 gtggcccttcccacagcgtcccgtagtacccttgccatggacaccacaggacttcctgca26640 cctggcatgctgcccttttgccatctctgggtaccggtgaccctagtggctgctggggct26700 gcacagcctgctgcctccatggtcatgttcccccatctcccagctctgcaccaccattgc26760 ccccacagccaccgcacgtcacagtacatgccagctagcgatgggccccaggcgtaccca26820 gactacgcagaccagagcacatagagggcagcatttgggcagaatatgtgctggtcaata26880 aatgtgtcagaaaatgagtaattttctgactgcacaaaaaagtcttcatggtctccatcc26940 tttttctttctttgcagtccagtacccccctccgacatctttggcttgcatgatagagac27000 ttcagcctaacacgcctccctggaaaagtgcccaggagatactcgagcaggtgctgggtt27060 ttaggaagcccctcagacatcccgcctctactcattaaaaattacctggagctggccggg27120 cgcagtggctcatgcctgtaatcccagcactttgggaggccgaggcgggtggatcacgag27180 ctcgggagatcgagactgtcctggctaacacggtgaaaccccgtctgtactaaaaataca27240 aaaaattagccgggcgtggtggcgggcacctgtagtcccagctactcgggaggctgaggc27300 aggagaatggcatgaacccgggaggcggacgtgcagcaagccgagatcgcgccactgcac27360 tccagcctgggcgacagagcaggactccgtctaaaaaaaaaaaaaaaaaaaaaaaattac27420 ctggagttgctcgctgagtgctaattgtgctgctagcattgccggcctagggttcacaat27480 gactctgtgttactggccattccttgtcatggcggtgcctttatataacaggatcttcac27540 tttacatacagatgagatcacgtctggctttgtcattgggttaaaaagtgcccttgatag27600 ttgtggaggtgacacatactaccgtcgactcgaatagatctgctaggcgccaagcttgtc27660 agccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggcaccagctc27720 aatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggactaaaaaat27780 gacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccag27840 aaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttcccacgttac27900 gtaacttcccattttaagaaaactacaattcccaacacatacaagttactccgccctaaa27960 acctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccaccccctcat28020 tatcatattggcttcaatccaaaataaggtatattattgatgatgttt 28068 <210> 17 <211> 30115 <212> DNA
<213> Artificial Sequence <220>
<223> Modified adenovirus <400>
aaacatcatcaataatataccttattttggattgaagccaatatgataatgagggggtgg 60 agtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaag 120 tgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgttt 180 ttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttg 240 tagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataaga 300 ggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgc 360 ggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcg 420 ttccgggtcaaagttggcgttttgattcggccgcttgggtcatagacttctttgagaacc 480 agttataagctatggtttctctccacagaaaaagcacttatggtgtctccccctttccag 540 cccaccaacattttacatctaatttgggggggttttctggaccacttaatacccatccat 600 ggatctcatgtgaagactcccctggcttgagaaatcactgtcttgttgaaaatgggaaca 660 aagctaagtcagatagctggttcatcagcaatgactttgaccaagcctgatcccacccta 720 ccccaccccaccccagtgaccaccccccacaatggagcacacaactctaaactggtttgt 780 aggtatgtgtgtgtgaacacgctgaggaatctgcaaaaccaaatggtgagtgcaaaacca 840 aacagtcacgagtaaatctcacaacaaccacgtcctgagctgcagcccttgttgaactat 900 accccactagggccccaagattttaggacttgtgtgtgggtgggacctcccctttctatc 960 atgctttagaagacagggatttcaccagaattgaacatattgaacatatgacccattttt 1020 ttcagccaaaggcaattaaaataacttcatacttgatatccatgtcagcaaaagctgcaa 1080 aacgcaaatgggtggctgctaagagccctggtaccctgacgagcacaccaagtgcttagc 1140 aacagtggtgtccaaaggaccagctggaagcctgccttgatgagaagttgctcttctttc 1200 tacatgaaggaacacctctactctcctgcttttaatacctgagctgtgagtgatcatcta 1260 tgtccattagcaaacatcccagtggagaaggaaacactcatacccgaaatctaagctaca 1320 tagttggaatcacttcaacttattgcaataaacacttactaagcacctattgtgggcaag 1380 tctttgcaatggataatagttcagtagatattttgatgtaatatttgaaataacaataaa 1440 aattgccaccactgaatttattgagcatttgctgtgctttaggcactaacccaggttctt 1500 taaatatttggtcttattcgatctgtataaatagccatctatgagaaagggactattatt 1560 gcccttattttacaaatgaggccaatgaggcccagagaggttaactaagttgcccaaaat 1620 catacagcccactagtggcagagcaggatgcaaacccaggcttgcctcgttcccaagccc 1680 acatgtcgtttgcattggtgtggaggtgtgcatgtgtttagtcattagcatgttatatga 1740 taagcaagttttgaaacatagaaacttaaaatgtgccattaagaaaagtacaggcaaggt 1800 tttccaaggggaggtgtggacctccggacaaatttttaagaactaattataaatacttaa 1860 aaatgggaataagaagacaacctaactacctgaacagttttagagatgactcatgcccac 1920 cctctaaaacccaaacaaaaacaacaaagtcaagaaaacccatgaaatcttagcaagcga1980 tttctatgtacttgtgaaaaggatttctttaccattctaatgggatttatgccaaccata2040 gagggctcagtgcccctcccatggggtggttagtgagtacagagctgagctcaccggcca2100 tctgcagcttcatgttatcaagctccagtttgtccttggagcaaggttatctgggacatg2160 agcagaggcattgctttctgcaatggacagttctttctgcctgcatacctagctccttga2220 taactttaaataccattttatagccacactggagttttgaagacctcaatatgcaaatat2280 tactcaggttctgattacttgtctgctccatgataacacactctaaaagcaatgaatggg2340 gcttatttgtagagaactgaagcattttaagcttttgctcaggaatccctggtagcttcc2400 tgtgacttgcaagatattagtgatgggtcaagaaacaggaccccccatcagcataacata2460 cgcagtgcctcagtagtccatcaggcagaaaaaactgcagatggcacatggaaatgacca2520 gcggcggaagataccccgacagtgtgggcagttctatttcagcagcaatcaagagggggc2580 ctggagccactcaatcaagtggagcaggatgggagcaagcactgtgcagaccaatgcaat2640 ccccagttaacacaaaaaataaataaaagagatgagattcagtctcttgactgtgactga2700 ctgggagctttatagctgatgcttgtgtcttttctccattttatttaattaggaaaagaa2760 atgcttatcacacactctacgtgtgaggtacaacctccacaggaaaggttgttaggaaca2820 tttcaacttctagaagtttctaaacataaggtaaatccatctttgtccttgggatcactg2880 cacatctcagaaaggcaaataaatcagtaattggtgggcataattactagctcatggact2940 gacaaggtctacactatttcgaatctcacagaagtaagccatgggacagatagagtctga3000 tagtggtgccccgtttcctggaggtcacacttactcatccccctggaccctgggcttctc3060 atgattgtcagagagtttgctggaaccaggtcagcccagtttcccttcccctgaaaaatc3120 ctccaatggctcgcaccaagactagagatgcaagtgcaagcacatccaccctctcagcag3180 ccaggttttgcgttccataatgtcacgtacccccagtcataccaatctccttggagctct3240 ccagacaggctgccatgtgtggtcggccctctgtgcttgtgctccttggtttgccaagcc3300 tggaatgccctttctccatgattgtactctgggaattctgtgtgtctttcgagatgaagc3360 tcctccaccctggaaattctgcctctctttccaggtccagctcctgtggctgaataccct3420 tggctgactgaacattcttgctgggccaagtcttaatcatctctaaatccctctggtgcc3480 ccgacagtatctagcccaggcaaagggctcagcaaatacttgcaaaattcaataaacttt3540 actatgttactagcttggcatgatgttctaggcacttgggagattataatctggtgagca3600 ttgtgctagtcttttttttgggataaacaccaatgataaactgagcctttactctcattt3660 tagcacatctactcatctcttttcaatgctggtgggtttataaaacccatcagtagaaca3720 gagagtgatcataatcatatggtgaaaataacatcagctaacatatttactgaacatgct3780 ttagtgtgctgggcactgaactctgtgcacatgtgaatttgagatagattgttcctagct3840 aataagatgaggaagtggagatgggcttactcaagtcacacaatagcaagatggggtaca3900 gacagaaaccaagctagaaaccaagcaacccctgggtttggaaatgcatgggctcctcct3960 ctgcacatggcgaggagcagtcaggtgctcctccttctctcatactgaaataactctgca4020 cttttggctattctgtgacactctgtgctattctgtagctcaaatggctctggtgcaagg4080 agctcagattatgactaacccccatagatattcagctgctttgcaagaagtggatgaatg4140 ctctggctttataaattattgactagattggatattggcccaatctccactctggtgatt4200 cggaaggaggcatatgcacatttgcaaggttatagtagtgcactctaattccactggctt4260 ctgagagcttgtaggtttcttgacttaatcattctggaattaaggtaatggatcctcaac4320 accttttctttccctggcctttactaaccatgtaacagaaatgagcagagaaaacccaag4380 aaagcgaactggagacttgatgagtgtgtcaaagatgctcagagtccaaggtcctctgtg4440 gctcactgacttcagaagccaacctccgttgttcaagtcacttgtgaggttactatgcta4500 gagcactaaatattatccagatagcccaaagaggtgaaggcagacatgtggaagaacctg4560 gatttttgggcaagttgatgaatgcctcctgccccatatcaaagagaggtgataggagac4620 aactttgtgaatttgaaataatgcaccggggaaacaggaaaacgtaatgtaagtcacgct4680 tcttggcttttttcttgccattaccctacttggccaagtgcaaatgggatttcaatatat4740 cataagtatgcatctattaataacaatgcaagaaagctgtacaactgaagtctgagattt4800 tgtaagaaacagaatctctgtaagcatcaccatccaacagaacttcctgagttgatgctg4860 aatcatcccagaaagaaggcgcgccagctctgcaggatcttcaagtctggggtgccacca4920 gcaagcgacggtcctccatgggctcttcaccttacggcagtgtccagaggcaccgccagt4980 cctctgctcctatgctggtcctgctgtccctggcaaaaggagccagagcattctctccag5040 gcctcccgaggaggctgcttcctttgttttgcagatggaggctcccatcctttgttctga5100 atcaatgtgctccaaagataagccccaagaaaacagttgttgccttttgacactgacaat5160 tagaatcgttggaaaatggagaaaacaggaaatggcaaatggtttcagtgaccaggagga5220 aaccgtgcctgaaagttgctgcttagtgactgggacactcgctttctgctctcttatgaa 5280 ggacagcctaggccgtgtggccttttataaacaaagctatgaaggggtcgtcaaattttc 5340 tagggctgcaactgtggcactacgtcctgttgtgccaggtgacactgacaagcagcactg 5400 agttctatgcaagcccaggtgtgcttctctcatggtgacccccagagaactaaggcccag 5460 ctcttcctctgtcacacccctcccagcccccactgtcagacaagggaccacattcacaga 5520 cagtctcagccaagatggcaaccttggaagtcctggggatgcctttctagaagctttagg 5580 ctgacgccagggagctgtaaagcccccacctgtccctgggggttgtgtgctgggcaggag 5640 agaggggaaagagcccaaactccaccactgcctgctggcacaactgagcccacgccaggc 5700 actgcaccagcttcactcaccagctaccacatgctaatgtctcctgtttacccaggtgca 5760 atcacctgagtgctgctttctcaccactgcatgggaagaacgatctcattaccaattcaa 5820 ccacttaagcaggcaaaaggactccaacgggggaaaggcagaggacaaacgattcgggga 5880 ctgagacagatgcccacaaaatgttcctggattttagccggattcgggctgaagttcctt 5940 gccctatgagcccttggaggagagcatcctgactcagacaagaattcaaatgacaagact 6000 tctgggggatggcagtgaatggatccaaagccttgttttataaagaatgtggaggaaggg 6060 aggaaggaagggagggaaggggagactgagagggagagagaataaagaagaggaagagaa 6120 ggaagaggaggaggaggaaggggaggggagaggaaaaaggggagggtagaggaggaggga 6180 gaaggggaagggaggacagaagaggggagggagcggggagattatctacccctccatgct 6240 agggaactcctgtctccttttccagcagacactgctccatgagtgcccccacagagagcc 6300 agcctgccacacagcagaaggtgctcagctatctgtcccaaaggttgagcactagtgtat 6360 accacaaggaaaaaaaccttcttttgtactgtttggaagccagaattagaaacaactcgt 6420 tcacccttctgactttccctttgcaatgacaggaagatagtatactgcatgggaccacca 6480 cacattcaagatctgagttgtggggacccccacccccacatgggtggagccccctgagac 6540 ccgtgggctctgactggccatggcccatagatagtgacaacatacacactgctatctgaa 6600 gcctagttggaggacggatggatgcttttaggaaaaaattccctttagtcgtccctcaga 6660 aactacaacggcttcctaaaatgaatggcaggcaagttttgattgtttcctaagttcccc 6720 ctaactctcagagcctgtgactcagcttctccagaatgcctgcagggaggcaggaccact 6780 gggagtaatgagcacgaattgggccacttctcccagtggactgttaacgccgagatctgg 6840 ggtggaacccactgaacacctgcccttttgcgttgcccagaatgcagccagagggatacg 6900 ggccacagtaactctggggcccctgaacctaccttcccaagaccctgacgggaaggtatt 6960 gcttacatgacagtccagtcagtccctctgtcactcagttaacaaacatggtcatgggtc 7020 tactatgggccaggctctgttccgggcatggggataacaccgtgtgtctctactcttatg 7080 gacacaagtaaatcaatcaaagcagataatgttctaatgaccacccagtagggtgaaggg 7140 atggagggtgggcaggtgctggcagaaacctaaggtcaagtcatgggaccccaatgggga 7200 actggaaactgccacaccattttctatgttaggggtctttccttgcccttctagagacct 7260 cacccatggtaatgatgctgccctcaggtttgaactggtctgaagctgtcactgggggct 7320 tcacaaaatgaatgccactgaaagcaaacggcagagatctgtatatgaaattctttttac 7380 cctttgttttctgtcttcccacatgatctatgactgctgcaaactctattctgactcctc 7440 ctcagtcttggccggtctgggaagaacttggtttagatgctgggaatgatcccgggcaaa 7500 ggaaacaaatatatttccagcttgcttttcttcttcccctgtggtgcctgcatcctctgt 7560 ctcgcactgactcacaggctggactgacaaagtggctgtgttgacagggaggatttagtg 7620 gatgccaggcctgaccctcaaaggccctcaggtggggccaatttccaaggttcttagaga 7680 agatttggggccacaaatctgctctgccacttcactgctgggtgaaagctgttggcttga 7740 gaagagagagaataggtgagggacaataattctcaaggcaagctagctgatatcctttca 7800 tttgtggcttccaaagacagggaaaggcaaggcagaccagggtggaaacagcgacatcct 7860 gaggggacgacgtccgttttgctaattctgtttccctggcagctttgtgctggacaagat 7920 ttctctggtttcagctgaagagtgaacagctgaggagctctgtgactctgagagagagag 7980 aaatcaatagcccttgaaaggaaagcactgcttgttcttccaggccagggcaggcaggat 8040 aaagagaacaaggctgtacataccaaagccgaatttccattaaaggccagaatcctggga 8100 ggccctttcagcttaatgaccatcttctgatcactatcacttagaaaatgttcatgtctc 8160 cagttttctctgcagatagtgcagacatcctgtgcacattgatattttatgcaccagatg 8220 caattctctgcaaaggtcccctctggctggtgtccatccgcttgctctcatgaccactga 8280 tgcgactctgggggcaagtcaagccattccttctagactctagaatctagaggatgatcc 8340 caactcacaggcctcttggacaagcttgctttggattccttctgcctgtactacccagac 8400 tgccagtcctccagcagggcaatccaacttcctgcctctagggtgtgggagtcctgggcg 8460 gggtttggaatccacagcagatggctggcaggagaggcagttcccgagcacccactaggt 8520 gccaagcaggcaggtatcaggtgcagggcttcggaggtgcagaagcacagcccctgtccc8580 cctcaggttcccagtctggcctcagggacagacaggcaggtaggtatggaaacggggtac8640 tggaaggcatacagctgagggagagagcagctgcaccctggtcaatcccccactcatttc8700 cctggaggtggggctgaggcacctggagcactctgcttgcttgggttgtccctttgccaa8760 agaatgccgggcagaaagtagagaacaagcaggcccaggaagcactagaagtccctcatg8820 ggtggacagagctttaccttcggagctgtcacctcctgtgttcctcccaacaaccctgtg8880 aggcaggcgggcaggctgagcaggggactgtgccccgttgacagatgaggaaagcgaggc8940 tcagggggaagtgacttacctgtggcgtcgcagctaggcagtagtggagcgggcacctgc9000 ctccaggccttcggatcccagctgccccagggcgctgcctctgaggtgctgtacgaggag9060 gtgactcgggccagctcatgagcaaaggagctgccgggcaggaaggactcactccccagg9120 gcctggcaggacgggggctgtcgcccgaggctgacctgggggtgcctggcttccccaagc9180 ttgcaggctttgtctacatgagtctacaacaaccgattgaacaatccatcagctgcttgg9240 ccacaaaatggttctgactgatcttgtcactgccctgagtgtctagctgggtcatctggg9300 tgttgtggttatacccaccaactgctttggtggaacgaaaccgcggccgatctgaattca9360 gatcggccgcgggatcattcctggactcagattgttctgaagaagcccagttctgggtgg9420 catcaagtgcttgctagatggggggcttgccttgatccggctacacttggaggtgacttg9480 ttcttggacggctacatacagaaagagagaagtggggatgagttccaaaggcatcctcga9540 cttcggctgtggccaccggagggtagctcctggcccaacacggacttctcacctcccgcc9600 cttggctctctactgagctcccccctgctccccaattcctcgccattcccctcatttctc9660 tgccctcagcctggactgcagttcttctgggaagctgccccaactccctaggtctgtgct9720 caccaagagcagatcacactggactgaaatgccagctgatttgtctcttcaagaaaattg9780 gaagctcctggaggtcagggtccatgtctgcttttacactcagtgctctgtatgcaggcc9840 tggcactgcccaccctttgacaggtggtgcatattttgtagaaggaaggaaggggccagg9900 tggggtgggctgggctggtggcgggagctagctcagcctcttagattctctacccgatgg9960 atgtgacctgggacagcaagtgagtgtggtgagtgagtgcagacggtgctttgttcccct10020 cttgtctcatagcctagatggcctctgagcccagatctggggctcagacaacatttgttc10080 aactgaacggtaatgggtttcctttctgaaggctgaaatctgggagctgacattctggac10140 tccctgagttctgaagagcctggggatggagagacacggagcagaagatggaaggtagag10200 tcccaggtgcctaagatggggaatacatctcccctcattgtcatgagagtccactctagc10260 tgatatctactgtggccaatatctaccggtacttttttggggtggacactgagtcatgca10320 gcagtcttatggtttacccaaggtcaggtaggggagacagtgcagtcagagcacaagccc10380 agtgtgtctgacccacccaagaatccatgctcgtatctacaaaaatgattttttctcttg10440 taatggtgcctaggttcttttattatcatggcatgtgtatgtttttcaactaggttacaa10500 tctggccttataaggttaacctcctggaggccaccagccttcctgaaacttgtctgtgct10560 gtccctgcaactggagtgtgcctgatgtggcactccagcctggacaagtgggacacagac10620 tccgctgttatcaggcccaaagatgtcttccataagaccagaagagcaatggtgtagagg10680 tgtcatgggctacaataaagatgctgacctcctgtctgagggcaagcagcctcttctggc10740 cctcagacaaatgctgagtgttcccaagactaccctcggcctggtccaatctcatcccac10800 tggtgcgtaagggttgctgaactcatgacttcttggctagcctgcaacctccacggagtg10860 ggaactacatcaggcattttgctaactgctgtatcctaggccaataaatgttgatcacat10920 ttatagctgccatggtagggtggggacccctgctatctatctgtggaggctctgggagcc10980 cctgacacaaactttctgaagcagagcctccccaaccccttttccattccctatacctga11040 cagatggcccaggaacccattagaaatggaaggtcactgcagcagtatgtgaatgtgcgt11100 gtgggagaagggcaggatcagagccctgggggtgtggcagcccccaagtgattctaatcc11160 agatcctagggttgtttccctgtcccattgaaatagctgctttaaggggcctgactcagg11220 gaaatcagtctcttgaattaagtggtgattttggagtcatttagaccaggccttcaattg11280 ggatcctgctcttagagttggatgaattatttaactgattttcagatctcctctttctca11340 atgctttcagaagcacagtaactgcttactctgaaatgaattctcaccccacttccacat11400 atgcaccccttgcccacccctttgggaacactggccttaactgcttaccttcaaatggac11460 tcatctgttgggagatatatgcattctgccgttcaggggtcattgccataagacctgatc11520 tctgttcctcttgctaaacagaagatgaaaaagacaaattagattacagctaccaattaa11580 taattagccttaggatcgctgcgtggggacctaggacttggctttggtgcagcagaaagc11640 atgaataaacacaccagcatacactcgcatgcatgccccaccctctcgagcaaaattcca11700 caggtataaataaagtaagattctgcacctgggttaaaaacacaactgcaacagcataga11760 atggggcaggagagacagaacttaatagcaagagcacacagaaaaaagttttaggcattt11820 tggatgtccatctgctcaggatgggtcagcagtgagatgcggtcaccaaaagaacaaatg 11880 taacattaggctgcattaatagaagcagagtatgtagaaggagggaggtgacagtcctat 11940 gctaactctgccttggccagactatacccacaggagtctgggcatgccagtctcagggag 12000 acccagacagactggctgcattcagaggatggtaagtaatgagagtggggattggacttc 12060 aaactacccagacaaagaatggctgagcaagccaaggatgctgtggctggggcagagcag 12120 actgtgggctatgtagtggtggatacctagcctctgcagggctgtcatagggaaaggaca 12180 ttgagaagaggactgaggcttgttcctggtggtcctggcatgaacggccagatgatcaca 12240 tggtcaggtggacacagtctccaacactgggagtagccaaacacttactgccaacctccc 12300 gcccttctcctgactagttgcagcataggcaattgggaggagcttcctgtctccatctga 12360 aagctggctgggtgggcagggggaggagcgagccaagtttcaaggccgcagtttcagcac 12420 tcagtctgggatcggctcaaggagcaaaggggaagaacatagccaggagggaataacatg 12480 aaggcccccagacccagaaaaggcatgacttgctctgagaccctcagccggttggtgtca 12540 ggttgtgactcggatccaggtctgactcccagtccagtgcttgaagcctcaccccacaca 12600 gtgaggggagcccggccatctctgctcaactgctgccatctctctccccttctcaaccac 12660 caaggcagctctgtctgggagcacaagctccaagtccactttctggtctgtgtccccccc 12720 aagatgccagaggacttgcctctacaacacgggctgcccgtgcagtgcctgcttttccag 12780 caaagggcttctgggaacccttctctgcactcagtggggctggtgggagtggggcggggt 12840 agcgacccagtgcttgggactgtgcccagctctcaggcctggcagcagttcctggccttg 12900 gttcctgccaaggcagagaggacaaacacatggcaccgggaagactacaccagaagcgat 12960 tccaccagactggggtttgcttttctatcccgcccttagcctgcttcctgtcctggtccc 13020 tgcctccccctccactggagctgccgtgtgggcagtgaggggctgtttctcagctgccct 13080 atggagctgccctctccctgccaaagcattggcaaggcggcaaggggtgggggtggggat 13140 ggggggtgggatctgccttctcaagctctcattatactgagcacgtctcacccattattt 13200 tatgtcatctagcaacaccccatgtggacactgaggagcatgggggtcacatgaccactg 13260 cccaaggccacaccatccggatctgcctgagatggtcagggttggcagccatttctgaag 13320 gcagtcctttcgctttggctcttcttgtaccagtctcaggacatcagggcagaagatcta 13380 cagtccccagcttactgatgtgacagcagaggctcagagaggttaaatgacttgcccaag 13440 gtgacacggctaagaagtacagtatctcctaactgcagaccaggtgcttctgctgcttct 13500 ggggacagattcctgcgtggctggctaggtctaaacggtccttaactccatccccaccgg 13560 ttgctgcattagtttcatcaaataacacagttgtacagaggtaggggttcaggggcaggg 13620 gcagatggaggctggagagtgtgactaaggaaacagcaggggaagtgcggtaaagtccga 13680 agggagggacggaaagagaaagccaagcccaggggcgtgccagacaaaaggaaaggccac 13740 gccggggcagggcaggcttcagcgggtgctggggcgtcttcatcccgggaagcacacatt 13800 ccagaggaccccggagtctaatggaaaagctggccagcctatcactatggaaactgccaa 13860 ggccacacagcgctacgcgtatttaaatgtgctggagtgttgagatactgtagtggtggg 13920 atgcttaagtgctggggtgctgggtgttgggatgcctaggtgctggggtgccgagatgct 13980 ggagtactagtgtgctgggatgctgaagtgctggggtcctgagatgctgcagtgctaggg 14040 tgctgagattctgggctgctggagtgttggggtgctgggatgctggagtgctgagatgct 14100 tggacaatggggtgctggaatactatggtgctggggtgctggggtattgagatgctaggg 14160 tactgggatgctgaagtgctgagatcctggagtgctgggctgctgggccacaggctcttg 14220 aatccattcgtctgcccaggggaagaaaccagaagataaagagctaatgaaggagctttg 14280 gttgagagggaggaagtaatggaaggagcaacatcttgtggaggagcaggagagaatgga 14340 cctcaggttgggagagagggccaggctacaggccagagaggcagaaggattccagcagag 14400 tgtgggctccaggagccaaggggaaacaggtttctgggaggagagagtccagtactgctg 14460 aagtggcaagtccgctgaggaccaggaagcttcatttggctttatgaccaggaggaattt 14520 ggaactgtgactagagtacttagggggaaggaggcaagactggagccagattgctctggg 14580 ttgaggggtgagtgggaggtgaagcagggcactgtcactcctttgaagggtggcagagag 14640 ctggaattggtgctggatgggctgtggggtgacagggtcatgtggaaagcccctgggggg 14700 cacctggaaaaggagaagctgacagtacagtgagaggacagctaagggaaagcggaatgg 14760 cagaacacgcactgccaggaggaatgaggatagggtcaggagtgccaggggcagtgaggc 14820 cagcctggggtcaggtggcaggacgtgtccaggaagctggtctgcactgcagcccacact 14880 ggctcagccttgaggttccctgtgtggttggggtaggaagttgaaccctctgggaatgga 14940 agatggaaccagctctgcgagccaagctcagcttttatctatgggtctctgagggctggc 15000 agagctgagtggggacaactgtgatccgtgaggctctcaggttgaggtggcccctccggg 15060 agggcttcattttcccagcgggtaggttctaagcagcagtggctgggcaggtgggtccaa 15120 cacagagccagagaagggtgaatgggcctcctggcaccccacccctgctgcccctgagct15180 cagtgatggagggggacagcacagctgagcccaagtgctttggtgtggccctgagggaaa15240 gctgcagcctgcctggggcctggcatggatgggacacttgaggcagagggacaatagtgg15300 gcgctgcagtgaggctggctcttggagaggtttcctgaggagtgctgcctgagacgggca15360 gggagaacagagacaaagttggtgacagggaatgaaagctgactgaaggactttacccag15420 acctatgaggatatctctctcagcaggaagcaggaggggactgtgtgaggactggccaag15480 agctggagtgttgggaaaatgactctttctccgacccctctgtcctagctctggcccctg15540 gactgcggaggtctgcttccacccccattggtcgatcgttgtcccttgtcacagccattg15600 agaattttggcagggagcatgttcttagagcatttttaggctctgcgggacataacagct15660 ctgcctcagagcacatgcctttctcagctcctgaaagccactgatcaaattggaacattt15720 tgtaccttagggatgaggatatcaactctcccagccacttagagggataaatgtgatgat15780 gcattcaattgtgactacatctgatcccaactgttgcttcagctgctctcctatagcaca15840 tggcgggaggcgtgcatcccagtagctacctccccacttttggggagatgtggttccatc15900 catgaaacctgggtacccgcctaccaggtcctggcctatcaggtggcagggtctggtcaa15960 agaagggcatgtgtggtcttcagcaagggagacaggacggtggtgcagagcgtctagacc16020 ctcagggcaagtctcccccacacctgctcccggggcagttgtctttgtgacctcccatcc16080 ccctctgtttcatcctctataaaatgaggggctgagccccaaaataacaggcttctttgc16140 catgatgcaaaactgctgaatctttctttctgacacacaaggcatcgagcagcctctgaa16200 agaaccaaagccactagcaggcttcctgacttgggtttgtaggtactgaatactcccttg16260 aaaaataaaaacatagaggcacttttctcctggctgtttattacagaacgaagaaaaaac16320 acactggcttgaaacagacgccagatttcaaatgtagaggtgaaatacgaggtggcaatt16380 aaaatgtgattacagaaagtctggacactgagaaaagtttacaggacagtgggtgtgggt16440 tttctataacagacacttaaatatacatgacgataattgcagatagaaaccatcaaagac16500 aaaccccaaatcaactaataatgtttacagatgttcccccccaaaccacagagccttaca16560 tcaaaacaaatactgaaaggctttaaaccaggaacagctcgccttaaccccacgagggtg16620 cacacaagctgggctttttctctcggtctgaatggtaaagggaggaggatactctagctc16680 ctccaggtggattgctgagacagggctcggctcacacactgtctctgcgcctctcccaaa16740 tctggagaactctcccagcctcctggtaaagtgtctctgtggggcacttaacgataaaac16800 agcttctgctgtaaagctcattaggaaagagctagcggagactgaaaggttcgcaaaaga16860 gattaagaatcacacaaggcaataggatttttagtgaacatagaaataaatggccaagtg16920 gttttctatttggcatttgtcaacttgcacaacaactcttggtcatatccacattgctca16980 ttgcattaaaaccataagcgactcagccacctagcttaacaaggtatcactggagcaaac17040 aacacggtctgcatatttgtaacattgtataataaacacaaaacaatgcatagtaaacac17100 aactctactgaaacaaaagccgtcgctttatttacaaagtcacaaaatgaagtataaata17160 cttctgtcattaatgtttaggaaaaccatttacaaaattttcaaatatgtacacgtagct17220 tgaaaaatcaccagctttccattttgtcacaggtagagagagggataagcatgggctgac17280 aacaccactcaaattgtaacgggagacaactgcgggtatggatcgacaccacttcctaga17340 gtgatgtcaccatgggggtttctatgggcatcctgctcagatttaaagtgccccagcatc17400 ctgggtgacttgcccagaattctgggctgtggcattttgagcagcagcatgctgttccaa17460 aatgtcgtcgatcagcctcaagttgcacacccagtcttcatctgggctcacacaggagcc17520 tttcaagagagcttcaatgaaatctacctcattgcagtcaggtgacgaaatcagatcatt17580 tagtgggggttggggctggcgcaaaaagtcggcaggtggcagctcagggggaatatccgt17640 tctgtcgaacggacctgggaactggctggcagcaacggcagaagcagcagcagcggtggc17700 agcagcagccacatagcttggtggctcgatgccctgtatggggctcaggggactaaagct17760 ggccataccctgctggaggaacttggtggtgtttgctacaggcaccgggccctgtaccgg17820 gctctgcctgaggctctggctgcccagcaggctgaagctggggttgttggccaggggcac17880 ttgtgttcccatcgcagcgggcacttgtgcctcccaatcagatggcctctgaaggcaggc17940 ctggccagaaggtgagtgctgctgaacgctattatccacttggctgaggggtgttttccc18000 cgaaactgctgtggtcacagctgctgccgctgtgacccatgcagcattgttgaacgcagt18060 gggcattcttggcacactaggccgtctgagctggtggggactcaaggactgggtgcccag18120 ggagctgggacagaacccaggcaggggcacttctggtggggtggccttggggctctgcat18180 atgctggcagacagagtcaagtctgcccaggggagtctggcctgagtgtgagaggatggg18240 acactgggggctggaggtgaaaattccttgccgcttccccagagttggtgagatcactcc18300 catgcccggcgcgcgcgtcgacgtgagcaagtggaatggagtggcagggaatgagtgagg18360 gagacaacagctcagtaggaccttgtgggctactggaaagactttggctttgactctcag18420 tgagataaggaggtatttgaaggagttttagcagagaaggaacaattctgtttattttta18480 ggagctctttatatatttaggaaattacttctttgttagtcatatattttggaatttgtt18540 gttccccgtttgtcatttctgtttctacattttgcttataatatttgtctatgtaaacct18600 taatatgtacttggttaaattgataaattctttgcataattaatagctagctattgttgg18660 cctacctataattaatttttgctttatctagtaataggcctgaattttcatttgggatcc18720 agccctctccctgacactatgcagggcctgggagactaaagcctgctggaaataactcca18780 gcttggccctgccaaacattacaacacctagcacctgctctccccaccatagtgactgat18840 ttgggcaggaatgagaccacagccactccaagagcccagctgttg~ctaccagtaacatc18900 ttctcctctaaacaggagatccagctatattgaaggcttccaaaattaccctatgtctac18960 ctgtcacaggaatgttcagaggactaagcctctgcctttctccaccccatacttacttca19020 tatggaactatgaatgggcaacttcacaatttattatttacgtcacctagatgcaagtca19080 gagggagaagcaacaaatcagcagtacctaagaagttctcaagtcctcaaggtgaacttt19140 acctatatatacaaaatatatttgcctctttcctttaaccctttttcttactcccaccac19200 tttccacccaatgtgtaataatattagtcacttcatagagttcttatgagaagttaaatg19260 aggaaatcatgtaaaccactgagggctggcaacaagttcttaataagtgctattgagtta19320 tgagtattattatctcccttgggctggattcacagtgtcacctcttctgggaccatagta19380 agccaatggttccagctccttctattatgtcattttaatttggggaatagcttaacatat19440 tagatgatcctttacatgcctgagcggtctttctttgatggtatttctatgatcttatga19500 agagctagatgttgccgctccattttatcttgctggtggcagctggtgtttagccaactt19560 gtctttttttttctgtctttatatttttggttttgttttttggcagaagcctgtgttttt19620 cagattactttttgcttgtgtctactttttcccaaaacaatattacatattaaacttatt19680 gtatatttaagtttattttatgcatcaaaagtcgcattgaatttctgtgctattgtgggg19740 caaattgacatcaatgtaatttaactcattttatcccaaattaggctatgtctttccata19800 tactcaaatcttcttttatgtccctcaacagtttccttcatataaatattgcatatttct19860 aattaagtttatttttctaatctccattttggatgtcttaaatatagtcttttctccctt19920 tgctctttatcaaagatctgcaaactatggcctgtttttgtaaataaagttttgtaataa19980 agtttaaatggccactcccatttgtttacatgttgagtatggctgctttcagactgaaat20040 ggcgaatgaagtggtcataacaagaccatatggcctggaaagcaaatatatttattatct20100 ggcaatttacaaaaaaagtttgccaacctctgctttatatttttgatctagttgttattt20160 gaaaataaagctattgatatttgtatattaattttttacccagctaccttattgttttta20220 ttacttaatagtttacagtatttcttttagtttttccaggtgtaacatctgtaaatatta20280 ataaatctattagctaatatttcatttagcatttttatatctatactcacatgagaagtt20340 ttctttaattttcttttgtgtttatcaacctgtgttcactcaggaaaatgaagccactta20400 agtataccaggagtggagggtttcatatagaaatgagggcttatttgaattttgggaaag20460 ctgatgaaacaggtcagcagagttactgcagaagaccaggaaagttacatctggaaggtt20520 agggaagcagacactaaagacttaagcctgaagcatagaagtaaggatccgtcaatactt20580 attgagaaacttctggaattctcaagaacctctgagaaatctcccattctgtggcaataa20640 agtggtggtactcaagccatcaccaacatggctcatatgtctcattgcactatgtccaaa20700 gacaaatggcctctgcttctcttctgtctcctaaatctcttaagatttcctcttatttgt20760 ttaagtctaacccagaagacttacatggcaggtaattctgggaaatgtattcccttgttt20820 ctccaaaggggtggggataataccaagttgacaacagaaaatctagcaccctttttaagg20880 ttttggcatcagtgatttcttggtgctctaaatagttcttatgattattccatgggtgct20940 taaaaagaatatgtattctctgatttaagaatatgtaattgatttaactattgattagat21000 attaataaaatagatttttaaattaccttattaatttggttaagtcctccatgactactt21060 attttttgactatttgttatggaaaggtgtagctaaataaatatagaactactctatatg21120 tgtttctagttctctatgtattctctacattttgctttataattgttttctttcttagag21180 acaggggtcttgctgtgttgcccaggccagagtgcagtggcatgatcataactcactgta21240 gccttaaactcctgggctcaagcaatcctcctgcctcagcttctcaagtagatgggacta21300 cagaagtgcatcaccacaccctgctaatttttttcttatttttatttttagtgatggagt21360 catgctatgatgctcagagtggtcttgagctcctggcctcaagcaatcctcccacctcag21420 ccccctgagtagctgggattataggtgagaggcaccatgcctggatttgctttataaatt21480 ttgatatgttacttggtgcatagatattcattgctgttagaccttcattatgtgttgtac21540 cctgcccttctttggttcacttttccttgaattcagtcttgtctgatattaattttcagc21600 tgtctgctttcttcttatttccatttttctaatatgcttttgcctactcatttgctttca21660 attttttctgagtcatttcattctagatgagtatattacagtcagtacttaattgggttt21720 ttctttgtcactgaatatgagtgtctcttttcctgtgtattgatagggtagatatgtgta21780 gtatgagtgttacaattttttaaaaaactttgatttctctcctttgttttatttggggtc21840 tccggttattttttaaggtgtatagtgtcattttatttttaatgtttgcctctgtaaaaa21900 taattttataggctgtacttaatcttctatttatttagctagtttatattgactcttcaa21960 taacagtagtaaatgtccctacttctaactccccttgccctccaccttttaattttgtgg22020 ggtatactactggccttagtttttctagtggttaattttatacatttaaataggtttata22080 ccttttaaacttcaatggccacctttaaaaatattttgaccccacccactataaaaaata22140 aacatgccatcttacacgcacaatctcactttctctccccttacattccctaatttttac22200 tgggtattttaatatttcttcatcatcgtggttgattacatttacatgtatattgcagtg22260 gtagttcccataattgttataacctttaaccatagttctacatttatttttgccaccgtt22320 tttcccattcaccttttggttcaactttatcttctagtagtgtcttcaaaaaagtgttct22380 tggaaactacagcccatgaaacattattgcatatttgaaaatatttatctgtcaccatta22440 cacctgaatgacaatttagctagctcaaatttactggctcacatcatcttttttttggta22500 attttttaagtacttctctactgtcatgtaatgttgtatgtaggatagcctaaagccaac22560 ttgtgtttttttattttgctgttatagtagtttgatcatttttgcctagatgccccatag22620 gattatttttctttgtctttatttcaggaaaccttactagactatggttcagtgctgact22680 agacatttttttccttaagaaacaccatgccctttagatccttagattcaaatctttttt22740 aatatcagtaaagtttttaaatttacatatttggatattttatacttctctagtttttct22800 tcttaaagaagaaaacattaattctgtctttgcctgtcttccatatctacaattttcttc22860 taatcactttttataatttccatttcattttgctcaattgcctcaaagctgctatctatt22920 tttctgttgtcaacaatctgtactcttacatcagctactatgataatattgtgtcaccat22980 ttactactatttacaagctaaccataatttctctgatgattttagttttctcttctactt23040 tgtgtcctagatacaggcagctcaattttaatttttgttgcttttcttgattccttatac23100 ctctgcattaagctcttaattcacaaattacttccattgttaagttattgagttcttaaa23160 caaatatttggtcatgattttcatctactccacatcaaaatgttcatactgtgtaggctg23220 tttgccttttttgtcctgctattttccaccattttgtgacatttttgtatggatctcctt23280 gttcgttttttattattagtcatccttgagtgagcagaaatcttcctagagcaggtattt23340 gaaagaagtagtgtggaagaggggctggggctgtgttctgcgttagtagaaaatccccta23400 attcttagaaaaatcctttatgattcatggtcttgtgggagaaggttgtgaagcgggttt23460 tgtgaatgtgcttcatgtgcaatagagagtgactgagaggtggtttaacctttcttctta23520 gctcacaaagaatggctgctacagagctgcttaacacccacagtttttcttctacatgct23580 gcacatttacaaagatggagtatccgtgagttttctcactcattcctacctcccctgcca23640 ttttaaagtatccaaggaagttggggagattccctcagctaaacagccacccagatctta23700 ttgttccaaacaaaaaattattagatttgccctctgagcaggtcacctactttggagaat23760 tttgtgctctgccagctctcctcaagatttggaaaagcagttattttctattggagcccc23820 catcactccagtccaaagttcactgcatgtgacatcccatcttatttgtctggttcctga23880 ttataggagtttcccagtttcaacaaaaatactgtctttgtttttgtttctcattcattt23940 ttccccaattttgtttcaaagcgaaaagtaaaaacattcttactctaacatcttaaaatt24000 aatgtctgggaggaacctaaactgctcattaatatatgccatgctatatgtgatatggca24060 gagactgctacctgtccaccagaatctatttatttttcttggatgcacaataagactaca24120 tttcctagtctcccttgcaattacatgtgaccatgtcattaagatttagtggatgaaatt24180 taagcagaagcaaggtgtgtaaattctagggctggcccattacaaactttaacatgggca24240 cctgtgtgtttttccttttctgcttaattgggttggagataatcacagtgatgctgaaag24300 ccatgtgctaaagaaggcagagcctccttctgcttgggctcctgaatgactgtgtggaac24360 agaggcccttgccactcttgggccattctggattattacatgcacaaagagtaaagttct24420 tttctgttaagccattagatgtatgggtttatttgctgttacagcctagtcgaacccaaa24480 tgatgtagtgagggaggcaggacaaggaagaaacctattcagtatggagttatccttagg24540 gacaagttacaaaaattataaggatcagacaccactgctggttgatccattatagaccag24600 aataagcaggtggtatgattccagaaggagatgccatggttgtgtgagagagagattaac24660 cattcctataaacagaagggtcaaagaaagtagccagtcaccagaatgcttatattaagg24720 aggattgtgactacctgtatataaagtataggaagaaaatggggcagggcaattaagatg24780 aattgcagacacaactatttgtgcatattaatttgaatttaaatttaaacttaatttaag24840 gataatttaaaggaaaatcttcccttacctatttcatgctgaaggggtctttcaagttgt24900 caaactctctctatataaataagactgcataatccagaccctccggaatccagagatgaa24960 ttgcatgtggtagctgatgtgtgggaagcccacagagcagcacttctggtcattgccatc25020 cagctagctggtgctctcagaagaatgagatgaagaggcatgactttcaagcttcctctg25080 ccatacaaggtgagtttgggttccaggcttcttcctagcccctgtttctgcttcctctga25140 tttaatacccagtcactgagtacctactaaacactcggcattaaactgaaaagaacaggg25200 aagtcaaaaaccttttaccaatgcctgatgggataaagacatgacccatccctcaaagag25260 cttgtggtcttcccatggcaatgcccttcctccctaggccagatggactgtgaagaacag25320 cagtctcaagtggatttattgggaagctcatgaagactcagctcaatgggccatgtttgc25380 aggggccccttacaaggctctgcacttgattttgtaattttgaattatttttctcaaaaa25440 ggcctcccaaattatgtaagcttcaggcctttaaaacttggatctgttcccttgaagcca25500 cataatctgatcagaaataattttcagggctcattgggtgctggagaattcatggttaga25560 tatttaaataaaaatgcagagaatctataggactttccaactgagaccaaggcttttttt25620 ttctggtagcttgactgccaggaaacagaaagcggggcatctccacctgctggcagaaga25680 ccaaaactacaccatattgcattgaccgcagacatcaggctttgtttccttttaagtgaa25740 taggtgaaatcaatcaagcaaactggaattaggaaacagaaggaccactcacaaccgtac25800 ctccacagcaaagctaacactttcattttgaataacccctttccagcccctgtttacatc25860 agtcacattttttgcatggacataatagtgtttcaggcagtcctgctctacagaaggcat25920 ttgtttcgctgaccataagcatcttatgtgcttctacattgtctttatagccagctgtct25980 gaagttctacatcatccgtcagtgatgggtcctgagtaaactcagcagtctcctctaagt26040 aacttgtatatatcccttttataaagaatgctgcagtggccacccttaagtatagaactt26100 tgtctttcccaggcattacttacaggaaactggggaccaaatgccactccatagcatagc26160 ttaggagaagtgagattccaaaggacagagtgggctggcacatatgtagtagaagactgg26220 acaaatgagagtgaatgagcgagtctgttgtgagtggatgacacgcaatgcaaccccaag26280 ggccggctccaatttgcacagtccctaaaacatcccagggatccagtcttatcgcagtct26340 catcaacattgacaataatcatttagagaattttcaccatgagacttttgacactctgct26400 ctgggattttggagttccaactgctttaacaatttatacagtcactcaaatgtagaatcc26460 agtgaattattaatgacaattttgctccgcatattgaaagggcattctatattaatcaat26520 tataaatcatgataaattgctttctaaaaattatacacttgaaacccagaaatgttcaag26580 cccctcaccagtttacatacatccttaaccgtgtcttgttaactgggtgaccaggaaaca26640 gagaaaggaggatgtacttaatgaataactggccagaagtcggaagacctagattttctt26700 cccatcccttccatagatttgctctgtgaccctagacaattcatatgccccctctgttac26760 catacttttaagctgaaggggttatagtgcatactttctgaggtccttctaacactggag26820 atctatgagttcataagatcacatagagtcttggacttttcagagatcactaggcataag26880 aaagccctcgaggacatcctcttcctagatcaaccactccaagctaagaccttaatgggt26940 ctacactgtcatccatttatataggagagaggatggtacaagctaagtgaaagtctgttg27000 tcaactcaggaactagggtacagttccagcaatcaatctccatggagggttagcagaggt27060 ctggcctagccaattagggtttggcactgccatatgctactagaaaacacatgctttcat27120 gccaggcaacagggtgggaagtgcaatgccattaaggaccataccttagcctacttaaac27180 tagcctgggattgagttactgagctcaaaatacaattcagcacactttcctgcccactgc27240 aagtcaggccagcccctggagaactccagggacaaggcaagtctggagtttgtccccaag27300 gaactcccagtctagtggaggagatgggcccatagcagagttacaaatgtccaaaataag27360 agcacaaattcctgggttcttaaagagagttcaagcacacacacacagagagagagagag27420 agagagagagagagagaaattaatttagataaggaagggaggtagggagagatggtgcca27480 gcaatggcttcctgaggaaatgtcacttgtgatgggtcttttaggtgagttggacattga27540 gagaaagtaatcaaaagggcacaagatagggtagctgtcaggtaaacaaaacaaaactgt27600 atgcacaaaggtctagaggcaagaaagcatgggtgtgcttgcatagcaatgaagaatgtg27660 gtttggccaaagcataggattcacagaaggacctccccaggccatcagtagtctctcctt27720 cgcaggagtccactgtggctggacgaggaacagatcaaaacaggtcttgtttgaggcttg27780 gcccaacaacagggccagctgttgaccagggcttccttcacccttagctttctctccccc27840 tagtgtagtcagagactccaaaggcagtcccagagtgttgtgccaaaacaaatgcacatc27900 aggctattgtcatcaggagcccttgtcttgttctgtttgttttaagaagacataagtgaa27960 ataaccaggtttccgaagagagggcagaattgggtttgtccccagggaaaatgaggtttc28020 tccaccatggctgttctccccatgcagcagcggccaggggtccccagcagctctttgctc28080 agtgcccctgatgctaggcctgatgcttcctatgtggtcagctcagatgtgcaacccttc28140 tccatttttattatttgagcaatataatcattattgcttttgttacccaaaatcttataa28200 tcacccatttaagagtaatgtaggaatcatttttctcttttatttgatgaactaatcttt28260 ctggttggctcttgaggtgggaggggtgggcaatggggaccagcctgcttcatctcactc28320 tttcctaggcctgctccttaggcacgtgccccacaaatcccacctccgcaggttggaaaa28380 ggggcccacagaggatctcaggtatctgaggaagggccagaggctatttggcctgacatc28440 ctggaacatcaccagccaggagccataactcctttaggctagcctgcctgcaactggatc28500 aggtatagcaaaaacgattgatctcaccttctaattttcaatgagtgggagtggctgcct28560 tgagtgctgtgttgggaaggagtctgaggcagcatctggaccctgtgtaaatatgcgttt28620 cctgttattgatgaagctttcaagacaagattttagaaagctttggctcaagttgtaggt28680 tgtgggaggaaggaattagtaagagccatcataaactcaacatctattgtgttccaggag28740 ctgtgaccactcccttctttttagtcgggaaggtcaggatctcactatgtcacctaggct28800 agagtgcaatggtgtgattattgctcattgtagtcttgaatttgaattcctgggctcaag28860 taatcctcctgcctcagccttctgagtaggtgggactactggtgtgtgccaccacactcg28920 gcttactcctccctttgtatcctgctgcatcgttagtgcatgatacccaccaccccctca28980 gaggcccagcctagtgctgtctctcagaaaaacaccctttgagtcaacagagacaaactt29040 agggggtgcctgactccaaagtccaggccttcctcctttgaggaacagcatggcttcccc29100 agggaatatcattccatggagacaaggcctgcagacagatctcattttcatctgttcttt29160 tactagcccttggaggatttgctccaaaaacctgaggcactgatgtttactccagcttta29220 cttcccccatacctggtgaacaactctggcccagcttccacacaccaaggcccatggctc29280 taatagagccagagaggcagtctcagctaagaagagctgtatcctccagggaccagcagg29340 aacttccagagatatcctacaagctccaactgctgggaaaagcagactggggtcttcctc29400 aggctccaccaaagatagccaagtggcagcaatgacatgagcacctcattcctgtcctag29460 accctggcaattccatgagagggaggctctgaattcacctctgttcccaggatgtgtgac29520 atcaatgcacattgtcataccagatacacctgccaaacttcagaacccctgggaagtgac29580 cagaatgccactgaattcaacaccttagaccaatgaacatgacctgggtcctgtaggata29640 ggaggcagcagcagtgtatacccagacctggtcgactcgaatagatctgctaggcgccaa29700 gcttgtcagccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggca29760 ccagctcaatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggact29820 aaaaaatgacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaaccta29880 cgcccagaaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttccc29940 acgttacgtaacttcccattttaagaaaactacaattcccaacacatacaagttactccg30000 ccctaaaacctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccacc30060 ccctcattatcatattggcttcaatccaaaataaggtatattattgatgatgttt 30115
FIGURE 3 is a gel showing restriction digests of helper-dependent adenoviral vectors grown with the helper AdLCBBHG and a helper of this invention.
FIGURE 4 shows the structure of different helper-dependent adenoviral vectors.
FIGURE 5 shows autoradiographs of labeled restriction fragments from helper-dependent adenoviral vectors. The figure illustrates the results of competition experiments involving groups of helper-dependent adenoviral vectors from transfection to passage 6. "A" is a competition of backbones containing the shuffled fragments of cosmid HUMDXS455A (C4). "B" is a competition of backbones containing part of the HPRT (hypoxanthine guanine phosphoribosyltransferase) genomic gene. "C" is a competition between 2 backbones one containing part of the HPRT genomic gene the other containing the shuffled fragments of HUIVIDXS455A (C4).
FIGURE 6 is a graph showing the correlation between GC content and helper virus contamination.
FIGURE 7 is a graph showing the balance between helper and helper-dependent adenoviral vectors at different time points after infection. The figure demonstrates the replication potential of different helper-dependent adenoviral vectors with the mouse Erytropoetin gene relative to the helper virus.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is particularly useful for obtaining and isolating helper-dependent adenoviral vectors produced using a helper virus. Co-cultivation of a helper-dependent adenoviral vector and a helper virus produces two different particles competing for packaging and results in a mixed viral population. The production of the helper virus in the mixed viral population reduces the amount of helper-dependent adenoviral vector produced, provides a viral contaminant that interferes with applications of the helper-dependent adenoviral vector, and produces a source of nucleic acid that can be recombined with helper-dependent adenoviral vectors As described herein, production and isolation of helper-dependent adenoviral vectors can be facilitated in different ways. One way is to employ helper-dependent adenoviral vectors containing elements favoring viral generation of the helper-dependent adenoviral vector over a co-cultured helper virus. Another way is to employ helper viruses containing elements that can be employed to reduce the ability of the helper virus to grow, and /or recombine with the helper-dependent adenoviral vector.
The term "vector" employed herein is used in its broadest sense, and is meant to encompass a linear virus which can carry a heterologous gene, and or a plasmid which can contain one or more regions of a virus genome. The term encompasses a helper-dependent adenoviral vector and a helper virus.
Adenovirus components present in a helper or a helper-dependent vector may in general be provided from any adenovirus. Preferably, a human adenovirus serotype is used; more preferably, human adenovirus serotype 1-47;
and more preferably, a group C serotype (e.g., serotypes 1, 2, 5, and 6).
Preferred group C
serotypes are human adenovirus serotype 2 or 5, and more preferably serotype 5.
A helper-dependent adenoviral vector preferably contains a heterologous expression cassette. Such a cassette is made up of a heterologous gene functional coupled to a promoter and regulator elements needed for gene expression, and is particularly useful for introducing nucleic acid into a cell. The introduced nucleic acid preferably encodes for a gene or is able to regulate gene expression.
Nucleic acid able to regulate gene expression include ribozymes and antisense nucleic acids.
The ability to introduce nucleic acid into a cell using the present invention has different applications such as in gene therapy, in antibody production (e.g., a vaccine) and research to examine the regulation of a gene in vivo or in vitro.
Different genes can be introduced into a cell such as Factor VIII, Factor VIX, CFTR, OTC, LDL, VEGF, FGF, EPO, HSV-TK, IL-2, IL-12, p53, HLA-B7, a-interferon, and cytosine deaminase.
A promoter is a DNA sequence directing the synthesis of RNA through an RNA polymerase. Suitable promoters depend upon the gene and intended use, and may include promoters such as EFIa, CMV, chicken a-actin (Arnold, et al., Nucleic Acids Res 1988 Mar 25;16(6):2411-29), and muscle creatine kinase (Johnson, et al., Mol Cell Biol 1989 Aug;9(8):3393-9 ).
-g-WO 00/46360 1'CT/US00/02405 Generally, the regulatory elements that are present include a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal. Regulatory systems are available to control gene expression, such as GENE-SWITCHT~'' (Wang, et al., Gene Ther. 1997 May;4(5):432-41, U.S. Patent No. 5,874,534 and International Publication WO 93/23431, each of which are hereby incorporated by reference herein) and~those involving the tetracycline operator (U.S. Patent Nos. 5,464,758 and 5,650,298, both of which are hereby incorporated by reference herein).
A helper-dependent adenoviral vector is preferably from about 28 kb to about 36 kb to provide for efficient packaging. An adenovirus can accommodate up to about 105°10 of the wild-type genome and has a lower packaging limit of about 75% of the wild-type genome. (See, Parks, et al., J. Virology 71(4):3293-3298, 1997, hereby incorporated by reference herein.) In a preferred embodiment, there are no bacterial plasmid-based sequences (such as an origin of replication or bacterial marker genes) present in the helper-dependent adenoviral vector.
Construction of helper viruses and helper-dependent viruses can be achieved based on the guidance provided herein using techniques well known in the art, such as those described by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A
laboratory Manual, 2°d Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein. Additional descriptions of useful techniques are provided, for example, in the Example section provided below and references such as Mitani, et al., Proc. Natl. Acad. Sci. USA 92:3854-3858, 1995; Fisher, et al., Virology 217:11-22, 1996; Kochanek, et al., Proc. Natl. Acad. Sci. USA 93:
5736, 1996; Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996;
Parks, et al., J. Virology 71 (4):3293-3298, 1997; and Schiedner, et al., Nature Genetics 18:180-183, 1998; each of which are hereby incorporated by reference herein.
The helper-dependent adenoviral vector and the helper virus can be generated in a wide range of host cells able to be infected by adenovirus.
Examples of host cells include 293 cells, 911 cells and PERC.6 cells (WO 97/00326 hereby incorporated by reference herein). The employed cell can be constructed to provide for useful proteins such as viral proteins or a recombinase.
HELPER VIRUS
Helper virus elements useful for facilitating the production and isolation of helper-dependent viruses include a low homology excisable packaging signal cassette and a substituted E3 region. Preferred helper viruses contain both a low homology excisable packaging signal and a substituted E3 gene.
Low Homolo~y Excisable Packaging-Signal Cassette The low homology excisable packaging signal cassette contains a modified packaging sequence which fulfills the role of an adenovirus packaging signal, has a low homology to the Adenovirus packaging signal, and is flanked by a 3' and 5' recombinase recognition sequence. Preferably, the modified packaging signal is less efficient than a wild type packaging signal. Advantages of such low homology excisable packaging signals include: (1) less recombination between the helper virus and helper-dependent adenoviral vector; and (2) when a less efficient packaging signal is used, less packaging of those helper viruses which escaped excision of the packaging signal.
Recombinase recognition sequences recombine when acted on by a recombinase that recognizes the sequences, resulting in the excision and circularisation of intervening nucleic acid. The recognition sequences should be sufficiently far apart, at least about 180 bp, to allow for the proper positioning of nucleic acid segments being recombined. Preferably, the recombinase recognition sequence is loxP or frt.
The loxP recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Cre recombinase. Cells, such as 293 cre cells, stably expressing Cre recombinase can efficiently remove nucleic acid located between loxP
recognition sites. (See, Parks, et al., Proc. Natl. Acad. Sci. 93:13565-13570, 1996 and Parks, et al., J. Virology 71 (4):3293-3298, 1997, both hereby incorporated by reference herein).
The frt recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Flp recombinase. Cells stably expressing Flp recombinase can efficiently remove nucleic acid located between frt recognition sites.
Flp-mediated gene modifications are described in U.S. Patent No. 5,564,182, hereby incorporated by reference herein.
Homology between a helper and the helper-dependent adenoviral vector encourages recombination events between the two, resulting in unwanted changes in the structure of the helper-dependent adenoviral vector or the helper virus, and leading to an increased contamination by helper virus. For example, if the first of a loxP site flanking the packaging signal is removed by homologous recombination within the packaging signal, the resulting helper virus escapes selection in 293cre cells because the Cre recombinase is no longer able to excise the packaging signal.
Sequences for different low homology excisable packaging signal cassettes can readily be designed taking into account recombinase recognition sequences and adenovirus wild-type packaging signal sequences using the guidance provided herein. The provided guidance focuses on the adenovirus serotype 5 packaging signal to illustrate the production of a modified packaging signal.
Such guidance is applicable to producing other modified packaging signals taking into account other adenovirus serotypes.
The wild-type packaging signal of adenovirus serotype 5 is formed by at least seven functional units called A repeats, which are located between nt 230 and nt 380 of the genome. The A elements have the consensus sequence ATTTGNBGC
(Schmid et al, 1997 J. Virol. 71:3375-3384, which is hereby incorporated by reference). The modified packaging signal of this invention preferably comprises less packaging elements than the wild-type (which has seven elements), preferably from about two to six elements, and more preferably from three to five elements. In a preferred embodiment, the modified packaging signal contains only four out of the seven original packaging elements (elements AI to AIV). The four elements are preferably in a modified form with two strong elements (AI and AII) present.
The position of the elements relative to each other may be changed, but in preferred embodiments, it is maintained.
In order to reduce contiguous sequence homology, the eight ambiguous nucleotides of the consensus sequence (ATTTGNBGC; SEQ. ID. NO. 1) within each A element are preferably replaced by sequences taken from a different A
element.
For example, the eight nucleotides within A1 were replaced by those from AV;
and the eight nucleotides within All were replaced by those from AVI. In addition, a new element was created between All and AIII starting 21 by after AII, by changing the existing nucleotides to the consensus sequence. Two more nucleotides were exchanged within AIV: ATTTTGTGTT (SEQ. ID. NO. 2) was changed to ATTTTGTTGT (SEQ. ID. NO. 3). One embodiment of a synthetic packaging signal is given in SEQ. ID. NO. 4.
Desired nucleic acid sequences can be produced using different techniques including those involving the creation of nucleic acid mutations and nucleic acid synthesis techniques. Examples of such techniques are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A laboratory Manual, 2°'~
Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein.
F~ Tnec~rtinn Homologous recombination between viral sequences (E1) present in the cell lines used for propagation of either helper virus (lacking El) or helper-dependent adenoviral vector, and the helper virus itself, has resulted in the generation of wild-type viruses. Homologous recombination results in the insertion of the region into an El-deleted virus genome. To avoid this problem, in preferred embodiments of this inventions, the helper virus also contains an insertion element.
Preferably, the insertion element does not contain a promoter or encode for a protein when present in the helper virus. More preferably, the insertion element is non-coding repeat-free human DNA devoid of splice signals.
Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996, describe the use of an E3 region containing an insert encoding for the ampicillin gene, the bacterial origin of replication, a CMV promoter and the luciferase gene (AdLC8BHG101uc). The insert protected against the production of viable viruses having a wild type E1 produced by homologous recombination. However, the insert impairs virus generation, most likely by interference with fiber gene expression. The resulting level of viral generation is disfavored for the production of viral stock.
The use of non-coding sequences as an E3 insertion element, preferably human non-gene sequences, provides for more efficient viral generation than that observed with E3 insertional elements encoding for bacterial protein described by Parks, et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996 (see Table 1, infra.) The use of intron sequences are preferred because such sequences do not contain hidden splice signals that could interfere with correct splicing of the fiber message.
The size of the insertion element (preferably between about 2800 and 3500 bp, more preferably about 2900 bp) is chosen to avoid the generation of potentially harmful wild-type viruses while maintaining a virus size close to wild-type size. Starting with a helper virus having a deleted E1, if homologous recombination occurs resulting in insertion of the El region, the recombined adenoviral vector which also contains the E3 insertion element would have a genome size of approximately 38350 by (108.1% of wild-type size). Because this size exceeds the packaging limit of Adenovirus 5, no viable virus would be produced.
The insertion element may be from virtually any source. In a preferred embodiment, a 2900 by insert was taken from human chromosome l 1q13, starting 13340 by upstream of STS marker 11S1337 (available from Genethon) and inserted into the Xba I site at position (nt) 28593 (referring to Ad5 wild-type).
The insertion element and its insertion point in E3 should be chosen to avoid any reduction in synthesis of the fiber protein. Such a reduction is likely to occur if the E3 region is modified because synthesis of fiber protein is dependent on correct splicing of RNA from signals located in this region. We demonstrate that viruses containing the insert grow to titers similar to those obtained with viruses containing an unchanged E3 region.
HELPER-DEPENDENT ADENOVIRAL VECTOR
Helper-dependent adenoviral vector elements useful for facilitating the production and stability of helper-dependent adenoviral vectors include an E4 non-coding segment that confers a selective advantage to a virus and stuffer regions) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. Preferably, the helper-dependent adenoviral vector contains an E4 non-coding segment conferring a selective advantage to the vector; and stuffer regions) providing the vector with a GC content of about 50% to about 60% and an overall size of about 28 kb to about 36 kb. In a preferred embodiment, there are no bacterial plasmid-based sequences (such as an origin of replication or bacterial marker genes) present in the helper-dependent adenoviral vector.
Stuffer DNA
Stuffer sequences are used to provide a helper-dependent adenoviral vector with an overall size of about 28 kb to about 36 kb. A helper-dependent adenoviral vector containing only a heterologous expression cassette, adenoviral ITR's and a packaging signal may be quite small, in general about 5-10 kb, depending on the size of the heterologous expression cassette. Because such a small virus does not package efficiently stuffer sequences are added to provide a final size of about 28 kb to about 36 kb. Preferably, the stuffer provides the vector with a GC
content of about 50% to about 60%.
Stuffer sequences used in gene therapy applications should not contain active genes or be recognized as foreign in a human cell. A preferred stuffer sequence replicates as well as the adenovirus itself, is not recognized by the host as foreign DNA and does not lead to chromosomal integration of the transgene.
Genomic DNA of mammalian origin, or even more preferably of human origin, are preferred stuffer sequences for gene therapy applications to avoid eliciting an immune response. However, stuffer based on human genomic DNA is identical to chromosomal sequences in a human target cell and could allow for insertion of the heterologous expression cassette into the host genome by homologous recombination. This phenomenon could have a potentially dangerous effect and should be prevented.
To prevent integration of the heterologous expression cassette into the host genome, the contiguous stuffer sequence can be interrupted, the individual fragments reversed and the expression units inserted at the specific breakpoints. (See for example, Figure l.) Using this strategy, the transgene is not flanked by regions which would support homologous recombination. Additionally, potentially unstable genetic elements such as retrovirus LTR as well as genomic repeats (e.g., MIR, and ALU) should avoided or removed to prevent rearrangements during amplification.
Another consideration is the GC content of the virus. The GC content that stuffer sequences provide to a helper-dependent adenoviral vector has a major impact on yield and purity of the virus. Competition experiments between different viruses as well as individual analysis revealed a correlation between the GC
content of a virus and growth properties. The GC content is preferably between about 50%
and about 60% and even more preferably between 52% and 57% which is close to the GC of wild-type adenovirus. Without being limited to any particular theory, advantageous growth properties are believed to be based on more efficient generation of a virus with a higher GC content.
Preferably, stuffer sequences are provided such that the heterologous gene cassettes) that are present are at least about 1.0 kb from the helper-dependent viral vector 3' end and 5' end, more preferably at least about 2.0 kb, and more preferably at least about 4.0 kb. Additionally, high AT rich segments such as those provided in the hprt fragment (59% AT) may be unfavorable even with a viral vector having an overall high GC content. A "high" AT is above about 55% AT.
E4 Non-coding Segment The E4 region of adenovirus type 5 occupies about 3000 by at the right end of the genome. Transcription from the E4 promoter is directed from the right end toward the center. Seven polypeptides are produced from open reading frames (ORFs) located within the region with the first ORF starting at nucleotide (nt) 406 from the right end of the virus.
It was observed that the E4 region is sometimes transferred from a helper to a helper-dependent adenoviral vector as a result of homologous recombination. This recombined helper-dependent adenoviral vector generally is favored in co-cultivation with the non-recombined helper-dependent adenoviral vector and dominates in vector preparations. Despite its strength, this recombined helper-dependent adenoviral vector cannot be used as a helper-dependent vector for gene therapy because it contains functional E4 genes. Expression of those E4 genes in a host cause an immune rejection of vector infected cells by the host.
In accordance with this invention, it has been determined that a noncoding segment of the E4 region confers a selective advantage when a virus is co-cultivated with a virus lacking the same element. The entire E4 region is not needed.
From adenovirus serotype 5, the part of E4 between the 3' ITR (starting 102 nt from the right end) and nt 405 which does not contain coding sequence is the segment that should be preferably included into the helper-dependent vector and is a non-coding E4 segment. E4 sequences located left of this element have no additional impact on virus strength. This is of importance because all genes (open reading frames, orfs) are located in the left part of the E4 region. Thus, another aspect of this invention is a cis-acting sequence located next to the 3' ITR (right virus end) which is preferably incorporated into the helper-dependent adenoviral vector because it supports amplification of the helper-dependent adenoviral vector.
Competition experiments were designed to determine the location of the beneficial element. Helper-dependent adenoviral vectors based on the vector stk120 (which contains only a 5' ITR, a 3' ITR and packaging signal as its viral-based nucleotides, as described in Morsy et al., 1998, Proc. Natl. Acad. Sci. USA
95:7866-7871, which is hereby incorporated by reference) were constructed containing either the 3' ITR only (stk120gfp), the 3' TTR and noncoding sequence of the E4 region until nt 400 from the right end (stk120gfp-E4promoter ) or the 3' ITR and the complete E4 region to nt 3115 from the right end (stk120gfp-E4). Pairs of these vectors were cotransfected at a 1:1 ratio to originate rescues of two competing helper-dependent adenoviral vectors.
After passage seven, viruses from at least three independent rescue procedures were purified, and DNA was extracted and analyzed by radioactively labeled restriction digests. The two competing helper-dependent adenoviral vectors have an almost identical restriction pattern. The only difference is in the size of one fragment, called the discriminating fragment. The discriminating fragment was found 5-10 fold stronger for stk120gfp-E4-promoter than for stk120gfp, and at least 4 fold stronger for stk120gfp-E4 than for stk120gfp (see Figures 2a and 2b). However, the discriminating fragments are of equal strength for stk120gfp-E4-promoter and stk120gfp-E4 in each rescue (see Figure 2c). Therefore an E4 element supporting helper-dependent adenoviral vector growth is present in stk120gfp-E4 promoter and stk120gfp-E4 and must be entirely located in the right part of the E4 region (between nucleotide -400 from the right end and the 3' ITR).
While not wishing to be bound by theory, it appears that there are two possible explanations for the selective advantage to the E4 segment-containing adenoviral vector: the E4 element allows for more effective replication of the virus or it confers improved packaging and stability. Our results indicate that the E4 segment does not effect replication. If infection is started with equal amounts of helper and helper-dependent adenoviral vectors, the ratio between helper and helper-dependent genomes inside the cell at the end of the production cycle (48 hours) is identical.
However, 3-fold more helper-dependent adenoviral vectors were packaged for the genome containing the E4 segment compared to contaminating helper genomes.
Thus, it appears that the E4 segment supports effective packaging.
EXAMPLES
Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
General Materials And Methods Construction Of Plasmids Cloning techniques described in J Sambrook, E.F. Fritsch and T.
Maniatis, 1989 Molecular Cloni~zg, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference, were used.
To generate plasmids containing the entire Ad5 genome, homologous recombination in E. coli based on the recF pathway was used. Competent cells of E.
coli strain BJ 5183 were transformed with a plasmid vector containing the adenovirus genome and a fragment from the shuttle vector. This fragment contains the sequence to be inserted flanked on both sides by sequences homologous to the vector. In order to select against re-transformation of the vector without modification, it was linearized close to the insertion point. DNA isolated from individual colonies of BJ5183 was retransformed into the strain XL2 (Stratagene) and high yield plasmid preparations were obtained. Correct clones were determined by restriction analysis and sequencing.
Virus Rescue 10~.g helper plasmid was digested with PacI and transfected by calcium phosphate coprecipitation into a 6 cm dish of subconfluent 293 cells.
Directly after transfection cells were overlaid with aMEM/ 10% FCS/ 0.8 %
Seaplaque agarose. 20-100 plaques appeared after 7-9 days. Plaques were taken days after transfection and used to reinfect cells. After two intermediate passages the cell lysate was used to infect NUNC-cell factories (NUNC). Cells were harvested 48 hours after infection and virus was released by three freeze/thaw steps steps (-70°C
/37°C). After treatment of the cleared lysate with BENZONASE nuclease, viruses were purified by ultracentrifugation on a CsCI gradient. After one step gradient (q=
1.5 / 1.35 1.25 g/cm3) the viruses were loaded on a continuous gradient formed from a CsCI solution of q= 1.35 g/cm3. Viruses were dialyzed against IOmM Tris pH
8.0, IOmM MgCl2 and 10% glycerol and stored in aliquots at -70°C. Helper-dependent adenoviral vectors were purified using substantially the same procedure. For analytical purposes virus obtained from two 15 cm dishes was purified. Virus DNA
was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using aP33dATP or dCTP and Klenow polymerise and separated in 0.7%
agarose gels. The gel was dried and exposed to film.
Determination Of Particle Titer lOp,l of virus suspension was added to 90,1 PBS/ 0.1 % SDS and incubated at 50°C for 20 minutes. OD260 is determined spectrophotometrically and the concentration of virus particles was calculated based on the equation 1 OD260 =
1.1x1012 particles.
Extraction Of Viral DNA and Restriction Digest 100.1 of virus suspension were added to 100,1 lysis solution (IOmM
Tris-HC1 pH 7.5, lOmM EDTA, 0.5% SDS, 0.05% pronase) under constant vortex.
After an incubation at 37°C for at least 2 hours followed by addition of 100,1 TE the lysate was extracted lx with Tris-HCl saturated phenol. Virus DNA was precipitated with two volumes of ethanol, washed extensively with 70% ethanol and resuspended in TE. Virus DNA was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using a,P33dATP or dCTP and Klenow polymerise and separated in 0.7% agarose gels. The gel was dried and exposed to film.
Extraction of Cellular DNA From Infected Cells 10~ cells are lysed inlOmM Tris-HC1 pH 7.5, IOmM EDTA, 0.5% SDS
and treated with 100~.g/ml Proteinase K overnight at 58°C. The lysate was extracted with Tris saturated phenol, phenol/chloroform/ isoamylalcohol (24:24:1) and chloroform, precipitated with two volumes of ethanol, washed extensively with 70%
ethanol and resuspended in TE.
End Point Dilution Assay 96 well plates were seeded with 1x104 293 cells in 100,1 per well 24 hours before the assay. Each virus stock was diluted 103 to 108- fold.
Dilutions were used to infect 24 wells each (50,1/ well). Positive wells were scored by cytopatic effect after 14 days and the virus titer was calculated based on two dilutions with intermediate numbers of positive wells taking the dilution factor into account.
Quantitative PCR
Real time quantitative PCR (ABI PRISM 7700) was used to determine the relative amounts of helper and helper-dependent adenoviral vector. Two specific target sequences were selected present either in all helper viruses (Ad5 sequences from 11358-11456) or in all helper-dependent adenoviral vectors based on stk120 (bp 12037-12176) (Morsy et al., 1998, Proc. Natl. Acad. Sci. USA 95:7866-7871). A
plasmid containing both target sequences was constructed and used as one standard for both amplifications. Within each target sequence, a set of forward primer, reverse primer and probe (which is located between the primers and contains a fluorogenic reporter and a quencher) have been selected. Separation of quencher and reporter generated a fluorogenic signal during the logarithmic phase of PCR
amplification which was plotted versus cycle number. The plot was used to calculate template concentration based on the software of the 7700 system (ABI).
Construction Of Helper Viruses Helper viruses for the helper-dependent system were made based on AdS. In order to obtain specific helper functions, the plasmid pAdEl-E3+
(containing the entire Ad5 genome with a complete deletion of the E1 region) was modified in two regions: i) between the left ITR and the promoter of protein IX; and ii) in the E3 region.
To introduce changes at the left end of the genome a shuttle vector "ploxOpack" was constructed. It is based on pUC (Boehringer Mannheim) and contains the following elements:
A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt 1;
B) a loxP site starting directly after nt 195 of AdS;
C) a synthetic packaging signal contained in the following sequence:
5'- GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA
TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC
GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA
ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG-3' (SEQ.ID.N0:4).
D) a second lox P site;
E) a linker containing cloning sites; and F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
For comparison, a second shuttle vector, "ploxpack", containing the wild type packaging signal instead of the synthetic one was constructed. It contained:
A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt 1;
B) a loxP site starting directly after nt 195 of AdS;
C) Elements AI-AIV of the wild-type packaging signal contained in the following sequence:
5'-GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG
GCGGATGTTGTAGT AAATTT GGGCGTAACC GAGTAAGATT
TGGCCATTTT CGCGGGAAAACTGAA TAAGA GGAAGTGAAA
TCTGAATAAT TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG-3' (SEQ.
>D. NO. 5) D) a second lox P site;
E) a linker containing cloning sites; and F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).
The second shuttle vector was generated by cloning the 4.9kb XhoI
fragment from Ad5 wild-type (AdSwt} DNA (nt 24797-29791) into pUC. A 2900 by fragment of human DNA located on chromosome) 1q13 starting 13340 by upstream of STS marker 11S1337 (Genethon) was obtained by PCR and inserted into the XbaI
site at nt 28593 (AdSwt) in either orientation. This fragment contains part of inton 2 of the human LRPS gene.
A strategy based on homologous recombination between plasmids in E. coli (Chattier, et al., 1996 J. Virol 70: 4805-4810, which is hereby incorporated by reference) was used to introduce those sequence elements into plasmid pAd5E1-E3+
containing a complete Ad5 genome with a deletion of El. The procedure allowed for insertion of those elements at exactly the positions described for the shuttle vectors.
The flanking Ad5 derived sequences were completely maintained. The following plasmids for new helper viruses were constructed:
pAdlspLI1 = Helper 14 -- has synthetic packaging signal, intron of LRPS in E3;
pAdlpLI1 = Helper 1 -- has wild type packaging signal, intron of LRPS in E3;
pAdlpE3 = Helper 11- -has wild type packaging signal, wild type E3.
Plasmids containing the Ad 5 genome with the described modifications were digested with PacI to release the virus genome from the plasmid and transfected into 293 cells by calcium phosphate coprecipitation. Resulting virus plaques were used to reinfect cells. Amplified virus stocks were characterized by restriction and sequence analysis.
1. Growth of helper viruses in 293 and 293cre cells Different helper viruses as well as a El deficient adenovirus (d170-3) (Bett et al. Proc. Natl. Acad. Sci. USA 91:8902-8906 which is hereby incorporated by reference) taken as control, were grown in 10 layer NUNC cell factories, purified in CsCI Gradients and dialyzed. The virus concentration (particle concentration) was determined by spectro-photometric analysis (OD260). 1.5 x 106 cells (293 or cre) were infected in a 6 cm plate with 100 particles per cell. CPE was apparent in all plates after 48 hours. Cells and medium were harvested, frozen and thawed 3x and the titer was determined in an end point dilution assay. The number of infectious viruses produced per cell was calculated and the results are shown in Table 1.
Table 1 d170-3 AdLCBBHGlOIuc AdIpLII AdlpLI1 Ad without Helper Helper 14 lox 1 Wt. 'IJ Synth. 'll 293cells 1200' 27 840 610 293cre4 cells1400 1.2 1.3 0.8 *infectious units per cell The amplification rate for all new helper viruses exceeds that of AdLC8BHG101uc (Parks et al., 1996, Proc. Natl. Acad. Sci. USA 93:13565-13570, which is hereby incorporated by reference) by at least 20 fold. The growth of the helper viruses in contrast to d170-3 is well controlled in 293cre4 cells.
These cells release only 1-2 infectious particles/cell. The rate of suppression is therefore dramatically increased for the new helper viruses. Viruses without or with the insert in E3 in either orientation do not differ much in virus yields; thus, the chosen E3 insert did not have a major impact on virus growth.
2. Amplification of helper-dependent adenoviral vectors over a single passage A characterized stock (known particle titer and helper contamination) of a helper-dependent adenoviral vector containing the human OTC gene was used to infect 293cre cells. Cells were infected with 700 particles of the helper-dependent adenoviral vector and 100 particles of one of the helper viruses per cell.
After 48 hours cells were collected, virus was released through 3 freeze/thaw steps and purified by Cs banding. Particle titers were determined by spectrophotometric analysis (OD260). Virus DNA was extracted and analyzed by restriction digests (Figure 3).
Overall virus yield was slightly increased for the new helper virus over helper AdLC8BHG101uc. However, the ratio between DNA-containing particles and empty particles is increased for the new helper. Since helper DNA is undetectable in the gel, helper virus does not contribute significantly to the overall output.
To determine helper contamination more accurately, quantitative PCR
based on TaqmanT"'' technology was used. Contamination was similar between the helper AdLCBBHGIOIuc and the helper LPLI1 containing the original packaging signal, whereas the amplification supported by helper LSPLI1 containing the modified packaging signal resulted in a lower contamination (0.29%) (Table 2). This is expected because yields of virus grown in 293 cells are lower than those of an identical virus containing the normal packaging signal. Therefore helper virus which would escape cre-selection should still have a selective disadvantage.
Table 2 HD Virus Helper Contamination Producti vit %cont. St. Dev. %RSD
AdLCBBHGlOIuc 4800 0.52% 0.1% 22%
Helper 1 (Wt. 5300 0.42% 0.12% 28%
'Y) Helper 14 (Synth.'I')8100 0.29% 0.06% 21%
3. Construction of modified backbones Constructions are based on plasmid stk120 (Morsy et al., 1998, supra) containing a CMV promoter driven gfp (green florescent protein) gene within the stuffer sequences at a unique SwaI site (stk120gfp). In order to incorporate the additional elements into the helper-dependent adenoviral vector, the 3'ITR
(130 bp) was extended to 400 by from the right end of the Ad5 genome. This fragment contains the complete E4 promoter region. However, no adenovirus coding sequences are present. The resulting adenoviral vector is stk120gfp-E4-promoter.
In a second construct, a 1353 by fragment from the right end of stk120gfp containing part of the stuffer sequence and the 3' ITR was replaced by a 3115 by fragment of Ad5 from a MunI site to the end of the genome (stk120gfp-E4).
This fragment contains the complete functional E4 region.
4. Competition between helper-dependent adenoviral vectors Genomes for stk120gfp and stk120gfp-E4-promoter were released from the respective plasmids and cotransfected with a circular plasmid containing the entire helper genome into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus at moi 1.
The mixture of viruses was amplified up to passage 7 where the viruses were Cs-purified. Five independent experiments were carned out. Virus DNA was extracted and analyzed for presence of the two virus types. In all experiments a 5-10 fold excess of stk120gfp-E4-promoter was found (Figure 2a).
This indicates the presence of a cis-acting sequence located next to the 3' ITR (right virus end) which supports helper-dependent adenoviral vector growth.
An identical experiment was carried out to determine whether the complete element is located within 400 by from the right virus end or sequences further upstream contribute to this feature. stk120gfp-E4-promoter and stk120gfp-E4 were cotransfected with the circular plasmid containing the entire helper genome (Helper 14) into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus 36 hours after transfection at moi 1. After passage 7, viruses from three independent rescue procedures were purified, DNA was extracted, digested with BsrGl and labeled. The discriminating fragments have equal strength for each rescue (Figure 2c). Therefore, the cis-acting sequence supporting helper-dependent adenoviral vector growth is located between nucleotide -400 from the right end and the 3' ITR.
Example 3 Construction Of Helper-Dependent Adenoviral Backbone Containin~lAlternative Stuffer Sequences The construction of the new helper-dependent adenoviral vectors (group A) began with the ligation of a 3498 by from fragment from pSTK120 (which comprised of the bacterial sequences origin of replication and ampicillin resistance gene, the right and left TTRs and the packaging signal) ligated with a multiple cloning site linker (SEQ. ID. NOs. 6 and 7) containing HindI>I sticky ends and cloning sites EagI, AscI, BgIII, NotI, Xbal, Mlul, EcoRl and Kasl. This clone is named "pITRF"
DNA from the region encompassed within HUMDXS455A (Genebank Accession # L31948) used as stuffer DNA was amplified from human genomic DNA
in four segments of about 4.5kb each using the following pairs of primers:
SEQ. 1D NOs. 8 and (PCR
9 1 ) SEQ. ID NOs. 10 (PCR2) and 11 SEQ. ll~ NOs. 12 and (PCR3) SEQ. >D NOs. 14 and (PCR4) PCR1 product was Klenow filled in and then digested with Ascl. It was ligated directly into the Ascl/EcoRV of pITRF to produce "Construct 1".
PCR2 product was digested with Notl/Ascl and cloned into the Notl/Ascl site of Construct 1 to produce "Construct 2". PCR3 product and Construct 2 were digested with Mlul/Notl (double digest) and ligated to produce "Construct 3".
The PCR4 product was first cloned into a TA cloning vector.
To avoid inefficient direct cloning for very large plasmids homologous recombination in E. coli BJ 5183 was used to insert PCR4 and additional genomic fragments. The technique requires a small shuttle vector containing the region which overlaps with the insertion point in the large plasmid. This shuttle vector was constructed as follows: 2 kb of the junction over PCR3 and vector sequence was amplified using Construct 3 DNA. The PCR fragment was Klenow filled in and then digested with Sapl enzyme and ligated into PABS helper-dependent-3.0, a small plasmid vector (KanR) cut with EcoRI followed by a fill in reaction using Klenow and a digestion with Sapl. Homologous recombination between Construct 3 and the shuttle resulted in Construct 4 (C4).
The helper-dependent adenoviral vector part of C4 is 20kb in length and has 3 unique restriction enzyme sites to incorporate 3 different transgenes (Ascl, Notl and Swal).
As a next step, a CMV promoter driven gfp gene was inserted into the NotI site of C4 by homologous recombination. For this purpose, a shuttle vector was built containing 2 kb of C4 overlapping the NotI site (pNot). The gfp expression unit was inserted by blunt end cloning into NotI and a fragment of this vector was used for homologous recombination with C4. Because a single NotI site is also present in the gfp expression unit, the gene can later be replaced by other genes using this restriction site. The gfp gene is located at a junction between 2 inverted genomic fragments which prevents insertion into the host genome by homologous recombination which would be likely, if the gene was inserted within a contiguous sequence.
To incorporate additional stuffer sequences and extend the 3' TTR to include the E4 promoter, a second shuttle based on pABSHD-3 was constructed containing 2 kb of C4 including the 3' ITR and unique KasI, SaII, BgIII and HindIII
sites for insertion. The E4 promoter was directly cloned as a Hind III/ SaII
fragment into this shuttle vector.
Stuffer DNA fragments AFO, HSU, ERl, and ER2 (Figure 4), were amplified from human genomic DNA by PCR Expand Kit (Boehringer) and cloned into the Topo-blunt vector (Invitrogene). AFO and HSU were inserted individually as SaII fragments, the ER1 and ER2 fragments were inserted sequentially after KasI/
BgIII and BgIII/ Sal digests. Recombination in E. coli BJ 5183 lead to the final backbones C4HSU (SEQ. >D. NO. 16), C4AF0 (SEQ. >D. NO. 17), and C4ER
(Figure 4).
Construction of the new helper-dependent backbones (group B) was started by replacement of a 8181bp fragment from stk120 by the gfp expression unit.
For this purpose a shuttle vector was built based on pUC 18 containing lkb 5' to the unique SwaI site and lkb 3' to the unique EagI site in stk 120. The gfp expression unit was inserted into the Bam HI site of this shuttle as a BgIII fragment.
The replacement was carried out by homologous recombination in E. coli BJ 5183 between STK 120 linearized with EagI and a fragment of the shuttle vector.
This step removes a part of the hprt region from the vector which was most frequently lost during adenoviral vector propagation. This vector "STKSEgfp" is 19.4 kb in length and misses about lOkb of genomic sequence required for propagation as a helper-dependent adenoviral vector.
Fragments AFO and HSU were cloned after SaII digestion into the Sal I site of shuttle vector pSHI-3ITR which contains part of the HUMDXS455A -fragment in STK120 and the 3' end of Ad5 starting from nt -400 containing the promoter and the 3' ITR. The fragments ER1 and ER2 were inserted step by step into the same vector as SaII /BamHl and BamHl/BgIII fragments. The 3 resulting different shuttle vectors were recombined with STKSEgfp linearized with PacI
to generate HXAFO, HXHSU and HXER (Figure 4).
Example 4 Generation Of Helper-Dependent Adenoviral Vectors Cells from subconfluent plates were washed with PBS, trypsinized and seeded to 6 well plates (Costar) at 3 x 105 cells per well in 1.5m1 medium.
Genomes for helper-dependent adenoviral vector were released from the respective plasmids by Pme I digestion and 2~.g of DNA were cotransfected with a 2~.g circular plasmid containing the entire helper genome per well. A 24 hour transfection was followed by infection with helper virus at 1 moi. Forty-eight hours later cells were lysed by 3 freeze/thaw cycles and the lysate was used to infect 106 293cre cells together with Helper virus at MOI 5. Cells were lysed 48 hours later.
The same procedure was repeated with increasing the number of cells until passage 6 at which two 15 cm plates are infected. At this stage helper-dependent adenoviral vector is isolated by CsCI banding and analyzed by restriction digestion.
Passage 6 is also used to infect a large scale preparation (1x10 layer Nunc cell factory).
Example 5 Competition Between Helper-Dependent Adenoviral Vectors Virus rescue in a competition experiment was performed using the procedures described above and, instead of DNA from a single vector plasmid, a equimolar mixture of 3 vectors was used. Three mixed rescues were carried out:
one with HXAFO, HXHSU and HXER; one with C4AF0, C4HSU and C4ER; and the third with HXHSU and C4HSU. No rearrangements were observed in either the individual or the mixed rescues.
As shown in Figure 5 there are only moderate differences between backbones within the first 2 groups with a slight dominance of the C4HSU
construct.
However, in a competition experiment between HXHSU and C4HSU the latter clearly dominated. Thus, the remaining hprt sequences in HXHSU have an adverse effect on adenoviral vector propagation.
Example 6 Analysis Of Large Scale Preparations Of Helper-Dependent Adenoviral Vectors STK120-EFIamEPO. C4AF0-EFIamEPO and C4HSU-EFIamEPO
To investigate the behavior of the new helper-dependent adenoviral vector in vivo, the gfp gene was replaced in STK120, C4AF0 and C4HSU by the in mice immunologically inert mEPO gene. Replacement was carried out using the shuttle pNot by homologous recombination. Viruses were rescued as described above. Large scale amplifications were carned out in Nunc cell factories and the virus was purified as described above.
Since the size of the helper-dependent adenoviral vector and the helper differ only slightly, the purification procedure does not remove helper virus from the preparation. Such a physical separation was not intended to allow for a replacement of gradient based by chromatography based purification in large scale. DNA was extracted from purified virus and the content of helper virus relative to the amount of helper-dependent adenoviral vector was determined by TaqmanTM quantitative PCR.
We observed a correlation between the helper content and the GC
content of the helper-dependent adenoviral vector (see Figure 6). The experiment with Construct C4HSU has the lowest helper content (about 0.18%) compared to a helper content of about 1 % for stk120.
Example 7 Analysis Of The Replication Potential Of Different Helper-Dependent Adenoviral Vectors Cells were infected with 100 part/cell of helper and 300 part/cell of the helper-dependent adenoviral vector. Three hours after infection 1/3 of the cells was harvested and washed with PBS. Another portion was harvested at 42 hours and a third fraction at 48 hours. Cellular DNA was extracted as described above and the relative amount of helper and helper-dependent adenoviral vector DNA was determined by TaqmanTM PCR (Figure 7).
Figure 7 shows that if the rescue is started with an excess of helper-dependent adenoviral vector, the ratio of helper-dependent/helper is maintained and even slightly increased for C4HSU, whereas it is strongly decreasing for STK120 and slightly decreasing for C4AF0. This shows that the nature of the backbone has a strong impact on the speed of replication. Whereas the Adenovirus genome coevolved with the adenovirus polymerase, the helper-dependent adenoviral vector sequence was artificially selected. Therefore a less efficient replication is expected.
However, the most important determinant seems to be the GC content of the helper-dependent adenoviral vector.
Example 8 Sequence Information The different sequences for the SEQ. ID. NOs. referred to in the application are as follows:
SEQ. ID. NO. 1 ATTTGNBGC
SEQ. m. NO. 2 ATTTTGTGTT
SEQ. >D. NO. 3 ATTTTGTTGT
SEQ. ID. NO. 4 GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA
TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC
GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA
ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG
SEQ. ll~. NO. 5 GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG GCGGATGTTGTAGT
AAATTT GGGCGTAACC GAGTAAGATT TGGCCATTTT
CGCGGGAAAACTGAA TAAGA GGAAGTGAAA TCTGAATAAT
TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG
SEQ. m. NO. 6 AGCTCGGCCGATTATTGGCGCGCCAGATCTGCGGCCGCTTCTAGAAACGC
GTGAATTCGGCGCCA
SEQ. ID. NO. 7 AGCTTGGCGCCGAATTCACGCGTTTCTAGAAGCGGCCGCAGATCTGGCGC
GCCAATAATCGGCCG
SEQ. ID. NO. 8 ATTGGCGCGCCTTCTTTCTGGGATGATTCAGCATCAACTC
SEQ. ID. NO. 9 GATCGTCGGCCGCTTGGGTCATAGACTTCTTTGAGAACCAG
SEQ. m. NO. 10 ATCAGTTAGCGGCCGCACAAGCTAAGATCACAAAGCTGTTT
SEQ. m. NO. 11 TATGGCGCGCCGCTGACACCCAGCCTGGGTGCCGGTG
SEQ. ID. NO. 12 TCGACGCGTAGCGCTGTGTGGCCTTGGCAGTTTCCATAG
SEQ. m. NO. 13 TCAGTAATGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTG
SEQ. m. NO. 14 TATTAAGGCGCCGGGCATGGGAGTGATCTCACCAACTCTGG
SEQ. ~. NO. 15 TCGACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGT
SEQ. ~. NO. 16 Helper Dependent vector C4HSU
The sequence is shown without transgenes.
AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG
AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG
ACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATG
TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA
CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT
GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA
GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC
TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT
TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC
GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC
CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT
ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC
TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG
GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG
CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG
AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA
GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA
AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC
CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT
CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC
ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA
TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC
CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA
GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA
AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC
TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA
ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC
TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT
ATTTTGATGTAATATTTGAAATAACAATAAAAATTGCCACCACTGAATTTA
TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG
TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC
CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG
CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC
TTGCCTCGTTCCCAAGCCCACATGTCGTTTGCATTGGTGTGGAGGTGTGCA
TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA
ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG
GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA
TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA
TGCCCACCCTCTAAAACCCAAACAAAAACAACAAAGTCAAGAAAACCCA
TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC
ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG
GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT
GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC
AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT
CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC
TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA
CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT
TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT
TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG
CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG
ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC
AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG
CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA
AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG
CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT
ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG
AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC
CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG
GCATAATTACTAGCTCATGGACTGACAAGGTCTACACTATTTCGAATCTCA
CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC
TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC
AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT
CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC
CCTCTCAGCAGCCAGGTTTTGCGTTCCATAATGTCACGTACCCCCAGTCAT
ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC
TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT
TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG
AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC
TGACTGAACATTCTTGCTGGGCCAAGTCTTAATCATCTCTAAATCCCTCTG
GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA
AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT
TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA
CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT
CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA
TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG
CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT
TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC
ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA
CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC
AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT
ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG
AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG
TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC
AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT
ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG
ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC
CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA
AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT
CCTCTGTGGCTCACTGACTTCAGAAGCCAACCTCCGTTGTTCAAGTCACTT
GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG
GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT
GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT
TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT
TGGCTTTTTTCTTGCCATTACCCTACTTGGCCAAGTGCAAATGGGATTTCA
ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA
CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC
CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC
CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC
TCCATGGGCTCTTCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCT
GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT
CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA
TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT
TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA
GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT
GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC
TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT
TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA
CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA
CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC
WO 00/46360 PCT/~JS00/02405 CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG
CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC
AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG
AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA
GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA
TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT
GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA
GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC
AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC
CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC
AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT
TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA
CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG
GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG
AAGGGGAAGGGAGGACAGAAGAGGGGAGGGAGCGGGGAGATTATCTAC
CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT
GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC
TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT
TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT
GACTTTCCCTTTGCAATGACAGGAAGATAGTATACTGCATGGGACCACCA
CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC
CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA
CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA
GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA
ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG
AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG
GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC
GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA
ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA
CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA
GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA
GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC
ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG
TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC
ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG
GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC
TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT
GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT
TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC
TCCTCCTCAGTCTTGGCCGGTCTGGGAAGAACTTGGTTTAGATGCTGGGA
ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC
CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA
CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC
TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG
GGGCCACAAATCTGCTCTGCCACTTCACTGCTGGGTGAAAGCTGTTGGCTT
GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC
TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC
CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT
CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA
AGAGTGAACAGCTGAGGAGCTCTGTGACTCTGAGAGAGAGAGAAATCAA
TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG
GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG
CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT
CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT
CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC
CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG
GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC
CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC
TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG
GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG
GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG
GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC
CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA
CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC
CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT
GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA
GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC
TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG
GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG
GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA
GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC
CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG
AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG
CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG
CTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATCAGCTGCTT
GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG
GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA
ACCGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCA
GTTCTGGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCG
GCTACACTTGGAGGTGACTTGTTCTTGGACGGCTACATACAGAAAGAGAG
AAGTGGGGATGAGTTCCAAAGGCATCCTCGACTTCGGCTGTGGCCACCGG
AGGGTAGCTCCTGGCCCAACACGGACTTCTCACCTCCCGCCCTTGGCTCTC
TACTGAGCTCCCCCCTGCTCCCCAATTCCTCGCCATTCCCCTCATTTCTCTG
CCCTCAGCCTGGACTGCAGTTCTTCTGGGAAGCTGCCCCAACTCCCTAGGT
CTGTGCTCACCAAGAGCAGATCACACTGGACTGAAATGCCAGCTGATTTG
TCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATGTCTGCTTT
TACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCCTTTGACAGGT
GGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGG
CTGGTGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGT
GACCTGGGACAGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGT
TCCCCTCTTGTCTCATAGCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTC
AGACAACATTTGTTCAACTGAACGGTAATGGGTTTCCTTTCTGAAGGCTG
AAATCTGGGAGCTGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGG
ATGGAGAGACACGGAGCAGAAGATGGAAGGTAGAGTCCCAGGTGCCTAA
GATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCACTCTAGCTGAT
ATCTACTGTGGCCAATATCTACCGGTACTTTTTTGGGGTGGACACTGAGTC
ATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAG
TCAGAGCACAAGCCCAGTGTGTCTGACCCACCCAAGAATCCATGCTCGTA
TCTACAAAAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATC
ATGGCATGTGTATGTTTTTCAACTAGGTTACAATCTGGCCTTATAAGGTTA
ACCTCCTGGAGGCCACCAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCA
ACTGGAGTGTGCCTGATGTGGCACTCCAGCCTGGACAAGTGGGACACAGA
CTCCGCTGTTATCAGGCCCAAAGATGTCTTCCATAAGACCAGAAGAGCAA
TGGTGTAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTCCTGTCTGA
GGGCAAGCAGCCTCTTCTGGCCCTCAGACAAATGCTGAGTGTTCCCAAGA
CTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGTTGCTGA
ACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACAT
CAGGCATTTTGCTAACTGCTGTATCCTAGGCCAATAAATGTTGATCACATT
TATAGCTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCT
CTGGGAGCCCCTGACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTT
TTCCATTCCCTATACCTGACAGATGGCCCAGGAACCCATTAGAAATGGAA
GGTCACTGCAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAGGATCA
GAGCCCTGGGGGTGTGGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGG
GTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAGGGGCCTGACTCAGGG
AAATCAGTCTCTTGAATTAAGTGGTGATTTTGGAGTCATTTAGACCAGGCC
TTCAATTGGGATCCTGCTCTTAGAGTTGGATGAATTATTTAACTGATTTTC
AGATCTCCTCTTTCTCAATGCTTTCAGAAGCACAGTAACTGCTTACTCTGA
AATGAATTCTCACCCCACTTCCACATATGCACCCCTTGCCCACCCCTTTGG
GAACACTGGCCTTAACTGCTTACCTTCAAATGGACTCATCTGTTGGGAGAT
ATATGCATTCTGCCGTTCAGGGGTCATTGCCATAAGACCTGATCTCTGTTC
CTCTTGCTAAACAGAAGATGAAAAAGACAAATTAGATTACAGCTACCAAT
TAATAATTAGCCTTAGGATCGCTGCGTGGGGACCTAGGACTTGGCTTTGG
TGCAGCAGAAAGCATGAATAAACACACCAGCATACACTCGCATGCATGCC
CCACCCTCTCGAGCAAAATTCCACAGGTATAAATAAAGTAAGATTCTGCA
CCTGGGTTAAAAACACAACTGCAACAGCATAGAATGGGGCAGGAGAGAC
AGAACTTAATAGCAAGAGCACACAGAAAAAAGTTTTAGGCATTTTGGATG
TCCATCTGCTCAGGATGGGTCAGCAGTGAGATGCGGTCACCAAAAGAACA
AATGTAACATTAGGCTGCATTAATAGAAGCAGAGTATGTAGAAGGAGGG
AGGTGACAGTCCTATGCTAACTCTGCCTTGGCCAGACTATACCCACAGGA
GTCTGGGCATGCCAGTCTCAGGGAGACCCAGACAGACTGGCTGCATTCAG
AGGATGGTAAGTAATGAGAGTGGGGATTGGACTTCAAACTACCCAGACA
AAGAATGGCTGAGCAAGCCAAGGATGCTGTGGCTGGGGCAGAGCAGACT
GTGGGCTATGTAGTGGTGGATACCTAGCCTCTGCAGGGCTGTCATAGGGA
AAGGACATTGAGAAGAGGACTGAGGCTTGTTCCTGGTGGTCCTGGCATGA
ACGGCCAGATGATCACATGGTCAGGTGGACACAGTCTCCAACACTGGGAG
TAGCCAAACACTTACTGCCAACCTCCCGCCCTTCTCCTGACTAGTTGCAGC
ATAGGCAATTGGGAGGAGCTTCCTGTCTCCATCTGAAAGCTGGCTGGGTG
GGCAGGGGGAGGAGCGAGCCAAGTTTCAAGGCCGCAGTTTCAGCACTCA
GTCTGGGATCGGCTCAAGGAGCAAAGGGGAAGAACATAGCCAGGAGGGA
ATAACATGAAGGCCCCCAGACCCAGAAAAGGCATGACTTGCTCTGAGACC
CTCAGCCGGTTGGTGTCAGGTTGTGACTCGGATCCAGGTCTGACTCCCAGT
CCAGTGCTTGAAGCCTCACCCCACACAGTGAGGGGAGCCCGGCCATCTCT
GCTCAACTGCTGCCATCTCTCTCCCCTTCTCAACCACCAAGGCAGCTCTGT
CTGGGAGCACAAGCTCCAAGTCCACTTTCTGGTCTGTGTCCCCCCCAAGAT
GCCAGAGGACTTGCCTCTACAACACGGGCTGCCCGTGCAGTGCCTGCTTT
TCCAGCAAAGGGCTTCTGGGAACCCTTCTCTGCACTCAGTGGGGCTGGTG
GGAGTGGGGCGGGGTAGCGACCCAGTGCTTGGGACTGTGCCCAGCTCTCA
GGCCTGGCAGCAGTTCCTGGCCTTGGTTCCTGCCAAGGCAGAGAGGACAA
ACACATGGCACCGGGAAGACTACACCAGAAGCGATTCCACCAGACTGGG
GTTTGCTTTTCTATCCCGCCCTTAGCCTGCTTCCTGTCCTGGTCCCTGCCTC
CCCCTCCACTGGAGCTGCCGTGTGGGCAGTGAGGGGCTGTTTCTCAGCTG
CCCTATGGAGCTGCCCTCTCCCTGCCAAAGCATTGGCAAGGCGGCAAGGG
GTGGGGGTGGGGATGGGGGGTGGGATCTGCCTTCTCAAGCTCTCATTATA
CTGAGCACGTCTCACCCATTATTTTATGTCATCTAGCAACACCCCATGTGG
ACACTGAGGAGCATGGGGGTCACATGACCACTGCCCAAGGCCACACCATC
CGGATCTGCCTGAGATGGTCAGGGTTGGCAGCCATTTCTGAAGGCAGTCC
TTTCGCTTTGGCTCTTCTTGTACCAGTCTCAGGACATCAGGGCAGAAGATC
TACAGTCCCCAGCTTACTGATGTGACAGCAGAGGCTCAGAGAGGTTAAAT
GACTTGCCCAAGGTGACACGGCTAAGAAGTACAGTATCTCCTAACTGCAG
ACCAGGTGCTTCTGCTGCTTCTGGGGACAGATTCCTGCGTGGCTGGCTAG
GTCTAAACGGTCCTTAACTCCATCCCCACCGGTTGCTGCATTAGTTTCATC
AAATAACACAGTTGTACAGAGGTAGGGGTTCAGGGGCAGGGGCAGATGG
AGGCTGGAGAGTGTGACTAAGGAAACAGCAGGGGAAGTGCGGTAAAGTC
CGAAGGGAGGGACGGAAAGAGAAAGCCAAGCCCAGGGGCGTGCCAGAC
AAAAGGAAAGGCCACGCCGGGGCAGGGCAGGCTTCAGCGGGTGCTGGGG
CGTCTTCATCCCGGGAAGCACACATTCCAGAGGACCCCGGAGTCTAATGG
AAAAGCTGGCCAGCCTATCACTATGGAAACTGCCAAGGCCACACAGCGCT
ACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGTGGGATGC
TTAAGTGCTGGGGTGCTGGGTGTTGGGATGCCTAGGTGCTGGGGTGCCGA
GATGCTGGAGTACTAGTGTGCTGGGATGCTGAAGTGCTGGGGTCCTGAGA
TGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGAGTGTTGGGGTG
CTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGAATACT
ATGGTGCTGGGGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGA
AGTGCTGAGATCCTGGAGTGCTGGGCTGCTGGGCCACAGGCTCTTGAATC
CATTCGTCTGCCCAGGGGAAGAAACCAGAAGATAAAGAGCTAATGAAGG
AGCTTTGGTTGAGAGGGAGGAAGTAATGGAAGGAGCAACATCTTGTGGA
GGAGCAGGAGAGAATGGACCTCAGGTTGGGAGAGAGGGCCAGGCTACAG
GCCAGAGAGGCAGAAGGATTCCAGCAGAGTGTGGGCTCCAGGAGCCAAG
GGGAAACAGGTTTCTGGGAGGAGAGAGTCCAGTACTGCTGAAGTGGCAA
GTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAGGAGGAATT
TGGAACTGTGACTAGAGTACTTAGGGGGAAGGAGGCAAGACTGGAGCCA
GATTGCTCTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCAC
TCCTTTGAAGGGTGGCAGAGAGCTGGAATTGGTGCTGGATGGGCTGTGGG
GTGACAGGGTCATGTGGAAAGCCCCTGGGGGGCACCTGGAAAAGGAGAA
GCTGACAGTACAGTGAGAGGACAGCTAAGGGAAAGCGGAATGGCAGAAC
ACGCACTGCCAGGAGGAATGAGGATAGGGTCAGGAGTGCCAGGGGCAGT
GAGGCCAGCCTGGGGTCAGGTGGCAGGACGTGTCCAGGAAGCTGGTCTG
CACTGCAGCCCACACTGGCTCAGCCTTGAGGTTCCCTGTGTGGTTGGGGT
AGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGCTCTGCGAGCC
AAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGG
GACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGG
GCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTG
GGTCCAACACAGAGCCAGAGAAGGGTGAATGGGCCTCCTGGCACCCCAC
CCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGACAGCACAGCTGAGCCC
AAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGCAGCCTGCCTGGGGCCTG
GCATGGATGGGACACTTGAGGCAGAGGGACAATAGTGGGCGCTGCAGTG
AGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCTGCCTGAGACGGGCAG
GGAGAACAGAGACAAAGTTGGTGACAGGGAATGAAAGCTGACTGAAGGA
CTTTACCCAGACCTATGAGGATATCTCTCTCAGCAGGAAGCAGGAGGGGA
CTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCT
CCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCA
CCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCCATTGAGAATTTTGGCA
GGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCGGGACATAACAGCTCT
GCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAGCCACTGATCAAATTG
GAACATTTTGTACCTTAGGGATGAGGATATCAACTCTCCCAGCCACTTAG
AGGGATAAATGTGATGATGCATTCAATTGTGACTACATCTGATCCCAACT
GTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAG
TAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCATCCATGAAACCTGGG
TACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAAGA
AGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCG
TCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGT
CTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTATAAAATGAGGGGCT
GAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAAAACTGCTGAATCT
TTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCA
CTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAATACTCCCTTGAAAA
ATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTATTACAGAACGAAGA
AAAAACACACTGGCTTGAAACAGACGCCAGATTTCAAATGTAGAGGTGA
AATACGAGGTGGCAATTAAAATGTGATTACAGAAAGTCTGGACACTGAGA
AAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATA
TACATGACGATAATTGCAGATAGAAACCATCAAAGACAAACCCCAAATC
AACTAATAATGTTTACAGATGTTCCCCCCCAAACCACAGAGCCTTACATC
AAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCTCGCCTTAACCCCA
CGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTGAATGGTAAAGGG
AGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGACAGGGCTCGGCT
CACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACTCTCCCAGCCTCC
TGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCTGCTGTA
AAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGTTCGCAAAAGAGA
TTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACATAGAAATAAATG
GCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGT
CATATCCACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTA
GCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGCATATTTGTAAC
ATTGTATAATAAACACAAAACAATGCATAGTAAACACAACTCTACTGAAA
CAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAGTATAAATACTT
CTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCAAATATGTACAC
GTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGTAGAGAGAGGG
ATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGGAGACAACTGC
GGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCT
ATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGC
CCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCTGTTCCAAAAT
GTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTGGGCTCACACA
GGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTG
ACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAAAAAGTCGGCA
GGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGACCTGGGAACTG
GCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGCAGCAGCCACA
TAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGCTGGC
CATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCT
GTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGG
TTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCC
CAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTG
AACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCCCGAAACTGCTGTGGT
CACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAACGCAGTGGGCA
TTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCAAGGACTGGGTG
CCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTCTGGTGGGGTGG
CCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCTGCCCAGGGGA
GTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCTGGAGGTGAAAA
TTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCGGCGCGC
GCGTCGACACGACAGGGACATACTGGGAATGCGCCCTTGCCGTGGAGGC
GGGGACCCGGCAGCGCTACGTATCCAGCATCAACCTGTATCCAGCATCAA
CCCGCCAAGTTCACTAACTTGGTAGGGGTGAGGTTAGGGATCCTTAGGAG
CCCAGGCAGCCAGACTTTCTGGGGAGCCCATTCCCATTTGTGTTGCCAAA
GTACCCCCAGCAGGTTGTGGGAATGTTGCCTGTGAAGAGAGTCTGTTGGG
GTGAGATCTTGTGTGTGTGCACAGGGTGACAGTTGTGTCCCATTTCCCGGG
AAGCTGTGATGGCAGCAGAACCTAGAGGAGCCTGAGAGAGTGTGGGAGA
GTGGGCCTCTGGAAGAGTAGAGGCTGCGGAGCCAGGTGCAGGGCTGTCT
GTCACCCAAAGGAAGAGGGACTGATGACTCACTGAGCGTGTGTGTCCCCT
GGTGGCAGCAGGCCCCATAGTGAACATACCATACCTTTTCTGTCCTGAGC
GATGCTCCCAGCAGTCCTGGGAGATGGAACGGTCCTTATTCGGCTCACAG
GAAGGACCGCCTTAACTGGACAGACACAGCAAGGTGCTAAAGATGCCTTC
CATCAGAGGCCAGGTTGGAAGCTCTAAAGAGACTTCTCTTGCTGTTCTCTC
ACCCACCCCCAGGTTGTGTGTGTCCCGCTGTGGATTCTCATGTCCTTTCTG
TGCCTGGTGGTCCTCTACTACATTGTGTGGTCCGTCTTGTTCTTGCGCTCTA
TGGATGTGATTGCGGACAGCGCAGGACACACATAACCATGGCCCTGAGCT
GGATGACCATCGTCGTGCCCCTTCTTACATTTGAGGTAAGCGTTCCACGGG
AAGCCTCTTCAGCCCCTGAAGCTTGCGCTTCCCCTGACAGGATTCTGCACC
CCTAGAAAGGCAGCCTCTGTCCCTCGAGCTCACAGTGAGCCCACTCCAGG
AGAGGGGAGAGAACACAGCCATCTCCGAGAGGGAGCTTCGGTGAAAGGA
GAGCATCCTTCCTTTCTCTTGGGGGCAGCACGTGGGGCTGGCAGGGAGAA
GAGTGCACCTTTTTAGCCATGGTGCCTCTGTATGGCTCCAGTTTCCACTCT
GGGGAAAGCAGAGTGGGATGTCAGATTTGTGTATTGGAGTCACGTGGAGA
ATTCTAGAATGGGAGCTGTTGACTCCTTAGAACAAACACCCGGAGGAGTT
TGCCATAAAACTGCTGGCACTGGGAACTTTTCAAGTGGATAGGCTATTGC
CGAGCTCTGAAGAGGGACATAAAAGCTCATTTCGAGCTTTCCCCAGGGAT
AGGTGGTTTCCTGCCTTTTTCTGGCGGTGCTGATGTTCCCTCTTGTGGGAG
CTCACGCGGGGGTGGGGTGGTGGGGAGGAACTGCCTAATGAAGTCTGGCT
TCCGCCTCTGCCCATTTTCGGTGCTGGCATCAACCGGGACTATGTCTCTTT
CTTTAGATTCTGCTGGTTCACAAACTGGATGGCCACAACGCCTTCTCCTGC
ATCCCGATCTTTGTCCCCCTTTGGCTCTCGTTGATCACGCTGATGGCAACC
ACATTTGGACAGAAGGGAGGAAACCACTGTATGTACTCAGCATTTCAGAA
GTCCTTGGTGTGTGTCTGGGGGGGGACCAGGGGGTGGGGGGTGGCGGATA
GAAGTCTAGGAAGGGATGAGTCCCCGAGGGCCCCAATTTAGAAGCTTGTG
TGGGAAAGTGAGGGCTGAGGAAATTCTGGGACCTTCTAAGGGAAGGGCA
TGCCGTAACTCTGGTGTTCTGCTGGCCTGCACCGGGACTTTTCTCGCAGTG
CACGCTGCCATTTGAGGTAGAACCAGACACGGCAGGCAACCTCTCAGAGA
TCCCGTTCCCTCCTCTGCAAAATGGGGATCAAGACAGATTCTTCCCAGGCC
CGGGAGGGTTTGATGGAAAATCCACATCTCCCACCCAAACCTGGGATTCA
TCCTAGGTCCCTGTTGGCCGCTCTGCCTCCCCCATATCCTTGCTGCCATCA
CCCGAGTCTTGCCTGTCTTGCCTTGCTAACACTCTATTCCCCTCCACCTGCT
TGCTGAGGCAGACACTTCCAAAACGATCTCTGCAGAGGGTGCCTTCCTGG
CAAGGCTGTGGGCTCCATGGCACGGAAGCCCAGAGCATTGCCCTTCGGAA
AGCCAGTGGGTTTGGGGGCAGGGCCTCACTGCAGCCCAGCAGCCCGGGCT
GTGCTTGCTGTTTGTGCCTCTGCCCCCTACCCCGCACCCGGGAGCAGGGA
GGGCTTGCACCGAGCTGACACTCCAGTAGCCTACAGAGAGGAGTAGTGG
GACTGGGAAAGTGGCTTTAAGGTGGCTCCATGAGTTCAGGCCCCCTCCTG
GCCAACCCGTGCATGACTACCGCCCTCACGGATTCCAGAGGGTGACAGAA
ATCTTGTTCTTGGGTGGCACTGTCATCCATGAGTTTATCCTGGCTGGAGAA
GATTAGCGGAAGACACCGTAGTCTGCGCACCACAGATATTTTGAGACTCA
CTGGAGCAGTAGTTCTCAAATTTGGGCATCCAGCAGAATCCCAAAAGGGC
CAGGAAAAGGGGACCGCTGGAGCCCACCCTAGCCCGACTCAGTTTCTGGA
GGTCTGGGCTGGGGCCCGAGAATGGCATCCCTAACTAGGCCCCGTGGACG
CTGTCCCTGCCGGTCCGGGAACCCCACTCCAAGCACCACAGAGCTAGCAT
TTGCACTTCTTCCCCATTTTGGGTACTCAAGCCCTGTTCAGGCTTTGTGACT
CAGGAGTCTGGATAAAGTATGTTATGACATTGTAGGAGTGAAACTTCTTG
TTACGGAAAGAAAGTTAACAGGAAGGTCAGTTGAGCCTCGTGTGTGAAAT
AAAAAATTCTTATTTTTCAGGGTGGTTTGGTATCCGCAAAGATTTCTGTCA
GTTTCTGCTTGAAATCTTCCCATTTCTACGAGAATATGGAAACATTTCCTA
TGATCTCCATCACGAAGATAATGAAGAAACCGAAGAGACCCCAGTTCCGG
AGCCCCCTAAAATCGCACCCATGTTTCGAAAGAAGGCCAGGGTGGTCATT
ACCCAGAGCCCTGGGAAGTATGTGCTCCCACCTCCCAAATTAAATATCGA
AATGCCAGATTAGATGCCACTTCCGGGGACAGAGCTTAAGTGGACTGGGA
CGCACTCTCTCCGCCTTCCTCTGCCCCCTCGTTCACCCCGCAGACCAGAAC
CAGTACTGGAGCTGGGTCTCCAGGTACGTCCATCTCATGCCTTGTTTGCAT
CCAGCGCCTATCAGCCACTCACCACGACGGGACGCGGAAGTGGCAGGTG
ACGGGGGTGTGTGCCAGCAGATGCGGATGCCAGGAAGAGTGTGAGAACA
GGGGTGGGATTACCGTCTGTCTGGGAGGGGCTCCAGGTACCCCTCTTCCC
CGTCAGACCCACTGGGAGATGGCTGCTTGCCAGGCCCCCAGAAGGAACAT
CTGTCTATACGGTGCTGAAATCCCAATCAAAAGTATTGTTTAGAAATGTAT
TTCTCCACAGGGCTGACCTCCTGCAGCTCGCTGAGCACTCCCAGGTCCTCA
GCACTCCCAGGTCGTGGCTGGGGCAGTCAGTAGGAACTGTAACTATGTCT
CTGATGCACCACGTGTTTAGACACAGCACAGTCCTTTTTTCTGTTCCTACT
GTGGAAGTAGTTTCTCTTTGGGCATGCTGACAGCAGTTTTTCATAGCCTCA
CGGATGAGCCCTTTCTACGGGAGTGACTCCATGCTTGTATACAGAGTATTT
ATACAAATGTTTTAGCATCTTCATATGCGGTGTTAACCCCTAGTTCTGTAC
AGCATATTCTGTTCAAGTATTTTTTTACAAGCTTGTGCTGTAGGCACATGC
CTTCTGCTGCAGAAGTGGACGCCCGTGGCACACTCCCCCCCCCCCCCCGT
GGGGTGCCACGCCTTCATGGGACATTGCCACTTCTGCCCTGGAACTCGTG
CAGGTACGTAGTAGCTGCTACTGCCACAACGGCAACACCAAGCAAGAGA
TGGTCCATGCTTTTCTGACGTTCTCAGAATAGTGGCTAGCTTCAAACCTGA
CAAGCGCTGCTTGAAGCCGGAACACTAGAGAATGTTGCTGAGAGCAGAA
ACGGCCACGCGGGTCACGACTATGCGTGGGAAAGTCTCAAGCTTCCCTCC
TGCCAGCAACAAGAAGGCTTTGGAGTAGGCATGATGTTTTCACGTGTGCG
TGCCGTTTCTCCAAGCACTGCAGGTTCCACCGTGTGTCAGAGGCTGCAAG
TTTAACATCCTCCTGCCTGAAAACAAATAGGTCCTTTGCTGAAAAGAGGG
TAAAAAAAGAGCTTTGATCTTCTCAGCCAGGAGAAGAGGGTGGTGTTTTC
ACGCGGGCAACTGCTCGCCGGCCTACATGGGGTTAATTCAAGTCTGCTGC
GAGCACGACTCCGCCCTTGGCACTGGCCTCCAGCAAGCCCTGTTCTCTTTG
GGGTACAGGGGAACGGGATGGTTTAGACTTTCCTGCTCAGTGTGTAAAAA
ATGTAGCTAAAGCCACTATTTTTGCTCTCCTTAAGCTGTTCAATAAACCGG
TTCCTCATTTTACACGTGCATGATGTGTATCTTCTTTGCTGGATGGGCCAG
GAAACTGGAGTGGTCCTCTCAGCCAGCCTCAGAGGAAAGAAATCTCTAGC
TGGCACAGGCAGCCAGTGAGTGAGGCTGGCGGCTGCAGGGGCACAGCCT
TTAGAATGAGTCCTTCAGTGCACAGGTCCCAGGGTATACGGGGTAGTGGG
AGGAAGGAGGGGACGCCTCGCAGATGCCACTGTTGGCTGGGCTACACCTT
GCCACACTTGTTACTGCTTAGGAGGCTTTCTGGAGTGTTCCTTGGGTGCTA
CGACAATCTGCAGCAGACACTGTCCTTTCACCGCTCCTGGTCCTCGTTTGC
TCCCCAGTGATGTCAACAGCTGAGGACTGCTCACGCTGCAACAAAAGGCT
CTGCAGTCGCTGTCTAGCTTGCCCTAGTCGTCTCTAGAGTTCTGCCTGAAC
TGAAACTCAAGTGGGGTTCAGCTCATGACTTGTGGCAATTGACCAGGAAA
TTCACCAGTTGCTGTGGCTGGAAGGATTTTCAGTCCTGTGGGTTGTAACCA
GAGGCCACAGGTGGATTCTGCCTTAGGCTCATGAGATTTCCGACTTGCTGT
TGAAGAAAATGCCTTGTGAAGTGACAACAGTAGCTCTGACCCAACTGCCG
GTGCCTCGCTAGTTCCTATACGTCCCACTGGATCCTCACAGCCCCGGGAA
GCAGGTGCTACTACTCTTATCCCCGGGAGGAGACAGAGGCCGAGAGAGG
TTAAGTGACGTGCCCAAGTCACACAGCTCGGCAGCGGCCGGGTTGAGCAT
CAGCAGTCTGTTTGCAGACCCCTCACTGTCACCCCCTGAGCCAGTGCGCCT
TGGGCCCTGCGGTCAGGATGTCTCAAGCGTGGAGGCATCACCGGTTCGTG
GCAGTCTCTGGAAGGTCACTGAGCTCTGTGCCCAGAATCGAGTCGGGGGA
GTCTGTGCAGAGGTGGCCCTGTGTGTGGGGACAGTGTGTGACACAGACAC
TGCTTTGGATGGACACCTCTCCCGTGACCTCCTAGCATCCAATCCCAAAGG
AACAACTGTTGCAGAGATGGACCGCTGGACACAAACCCACGTGCGTTTCT
CTGGAGACACTGGCCAAGGAAAACAAAACATGCTCGAAGGCCAACAGCT
GCATGCCCCACCGCGATGTGACCGCAGACACCCGGGGTGTAGAAGGGTCT
CTGCCTGGTGGGGGGACACGTGCAGGCCGAGGAGAGGCAGGAAGGAGGC
TGCCTCCGACTCCCCACTGGACTGCATGGCGACGGCGTGTGGTGGGGCAG
TCAGCTAAGCCATTTGCCTAAGGGGCTGTCGGGCATCTGCGTGCTGGGGA
CCGACAGTGTGGGTGTGTTAGGAGGATCTGTATGGAGCACATTGCTGCCT
CTGGCTAGGACAGGGTGGAAAGGGTGGCGTGGCTACAGCCTGACCCATG
GGCACCGTCCTACCCTTTGTTCTGTGCTTCCGAGTGTCAGTCATGTGCTGG
GGTCTGTGGGCCCATGACTCAGACGGTGAGCTCTGACCTTCCTGAGCCAG
GGCTTTGCTGTAGTTGTGCCTGGCTCAGGAGCTCTAGGACAAGGGGACCG
CTCCAGGTCTGCATCTACGGTGTGGCAGGGCCCCTCGGCACTCTTGTGCAC
TAGTGTCATCTTTCCCATTGAAATGACTGTGAGGACCAGAATGTGCACAT
GCAGATGGGCAGCTACTTGTCTGCCTTGGCCCTTTATTACACAACTTGCTG
GGGGTGGAGATGCCACCCCCCGGCAGTCAGAGCCCCTTTATGATGTCATG
GGGCTGGTTACATGACTGCCAAGGGGTGCTGCTGGCCACACTGCACTAGC
AAGTTTGCCAGATGGAGGACAAGCGATCATTGAGTATGGCTCGCTGTGAA
GAAAGAAATTCGAGAGGACAGGATCATGGCTTGGAAAGGGTGCCTTTCCC
TCCCCAGTTGCAGTCAGAGACCTACCTTCACCCAGCAGATCCTTCCCCTGC
CTGGGACGACCCGGGGTCCACTGGGAGCCCTAACTTGAGGCTGCTGACAG
AAGAAATCGCTTTCCAACCTCTGGCCGAGGAAGCTTCGTTCAGAAGGCCG
CACCCTGACGGTGACGTCCCGCCCCAGGGAGAAGATAATCTCCTCTCCCT
CCCCTTTCCACAGAAACTGTGGAGACTGGTCAGCAGCAACCAGTTTTCGT
CCATCTGGTGGGATGACAGTGGGGCTTGTAGAGTGATCAATCAAAAACTC
TTTGAAAAGGAGATTCTCAAAAGGGACGTCGCACACAAAGTGTTTGCCAC
AACTTCGATAAAGAGCTTCTTCCGCCAGCTAAACTTGTATGGCTTCCGAA
AACGGCGTCAATGCACTTTCAGGACCTTCACCCGCATTTTCTCCGCAAAA
AGGCTGGTCTCCATCTTGAATAAGGTAATGAACGACAAGCCTCTGGAGGG
GTTAAGTCGGTGGGCTCTGGGGCCTGGTCGGGTGGAAGTCCCAGGACTGC
CTCCTGGGAAGTGGGCGACCTCAGGCAGGGTGTGGGGCCATCGCTGTGGG
CCTGTGTCCCCCTCTGGGTGGAGGTGACATGAACTAAGAGTGAATGTGGG
GAGAGGGCTGAGGATGGTGCGGGCCCCTCTCGAGTGTGTAAAATATCACA
GGTGCCAAGTAGCCGTATCTGCGTGTCGTCCTCCCCGGGGCCAGCCATGT
CATCTGGTGGTTGCTGTGTCCCCCTGACTCCACAGCACATTACCCTGTGAG
GTGAGCAGGCCAGGGGAGTCTGGTAT'T"TGTACCACTGTCACCCTAGCTGG
TGTCTGGAGAGGTGCTCAAGTGGAAGCACTGAAGGGCGCCTGGCGCAGG
AGGTGCAGATGCTCCTGCTGCCCTTGGTAGGTGGGCCCCTGGTGTGGAAG
AGCCAGTACCCAGGGCCTCCAACCCAGCCGGGGTGCATTCTGTTGCCAGC
TGACACTGCATGGGGGAGGCCCAGAATCTTCTTCCCTCCTGGTCTGCAACT
TCAAAGACCCTTTCCGCCGGCCATGGACACCCTAATCTGCCATTTTGAGGC
TTTTTCCAAGACGGAAAGGCCCGCCACAACTTGGTAAACCTTGACGATGT
GAACGCGAGTCCCCAGCTTCCTTTGGGGACTGGGACCTTTTCCAGAAAGG
CCTCCTGGGCCAGTAGAGTTCTCTTGCACAGGGGCGTAGATGGTTGGTAG
TTGTAGTCCATCCTTGTGACTTGCAGTTTCCTTTTGTTTATTTAAACTTTGA
CTTATAGTTAGAGTTCTACTGCCATCCTTACTTTCAAAGAGACTCCCCTCA
CCTCCTCGTGAGGATGAAGAGAAGAGTGGGTGTCAAGTCTGCACCAAGAC
ATCAGGAGGAGGACAAGCCAGAAGCTGCTGGATCCTGTCTGGCACCAGC
AGACACTGAGCAACAAGATCACACGTCTCCGAATGAGAATGACCAGGTC
ACACCGCAACACCGGGAACCGGCCGGTCCCAACACCCAAATCAGGAGTG
GCTCTGCTCCACCAGCAACTCCTGTGATGGTGCCTGATTCCGCCGTGGCGA
GTGACAACAGTCCAGTGACCCAGCCGGCCGGCGAGTGGTCAGAGGGCAG
CCAGGCTCACGTCACTCCGGTGGCCGCTGTCCCTGGGCCTGCAGCGCTGC
CCTTCCTCTATGTCCCTGGATCTCCCACTCAGATGAATTCTTACGGGCCTG
TGGTGGCCCTTCCCACAGCGTCCCGTAGTACCCTTGCCATGGACACCACA
GGACTTCCTGCACCTGGCATGCTGCCCTTTTGCCATCTCTGGGTACCGGTG
ACCCTAGTGGCTGCTGGGGCTGCACAGCCTGCTGCCTCCATGGTCATGTTC
CCCCATCTCCCAGCTCTGCACCACCATTGCCCCCACAGCCACCGCACGTC
ACAGTACATGCCAGCTAGCGATGGGCCCCAGGCGTACCCAGACTACGCAG
ACCAGAGCACATAGAGGGCAGCATTTGGGCAGAATATGTGCTGGTCAATA
AATGTGTCAGAAAATGAGTAATTTTCTGACTGCACAAAAAAGTCTTCATG
GTCTCCATCCTTTTTCTTTCTTTGCAGTCCAGTACCCCCCTCCGACATCTTT
GGCTTGCATGATAGAGACTTCAGCCTAACACGCCTCCCTGGAAAAGTGCC
CAGGAGATACTCGAGCAGGTGCTGGGTTTTAGGAAGCCCCTCAGACATCC
CGCCTCTACTCATTAAAAATTACCTGGAGCTGGCCGGGCGCAGTGGCTCA
TGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGAGCT
CGGGAGATCGAGACTGTCCTGGCTAACACGGTGAAACCCCGTCTGTACTA
AAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCACCTGTAGTCCCAG
CTACTCGGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGGAGGCGGAC
GTGCAGCAAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAG
CAGGACTCCGTCTAAAAAAAAAAAAAAAAAAAAAAAATTACCTGGAGTT
GCTCGCTGAGTGCTAATTGTGCTGCTAGCATTGCCGGCCTAGGGTTCACA
ATGACTCTGTGTTACTGGCCATTCCTTGTCATGGCGGTGCCTTTATATAAC
AGGATCTTCACTTTACATACAGATGAGATCACGTCTGGCTTTGTCATTGGG
TTAAAAAGTGCCCTTGATAGTTGTGGAGGTGACACATACTACCGTCGACT
CGAATAGATCTGCTAGGCGCCAAGCTTGTCAGCCTTACCAGTAAAAAAGA
AAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCAC
AGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAA
TGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGA
ACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGT
CACTTCCGTTTTCCCACGTTACGTAACTTCCCATTTTAAGAAAACTACAAT
TCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCG
TTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG
GCTTCAATCCAAAATAAGGTATATTATTGATGATGTTT
SEQ. ~. NO. 17 Helper dependent vector C4AF0 The sequence is shown without transgenes.
AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG
AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG
ACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATG
TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA
CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT
GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA
GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC
TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT
TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC
GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC
CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT
ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC
TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG
GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG
CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG
AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA
GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA
AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC
CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT
CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC
ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA
TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC
CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA
GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA
AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC
TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA
ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC
TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT
ATTTTGATGTAATATTTGAAATAACAATAAAAATTGCCACCACTGAATTTA
TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG
TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC
CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG
CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC
TTGCCTCGTTCCCAAGCCCACATGTCGTTTGCATTGGTGTGGAGGTGTGCA
TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA
ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG
GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA
TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA
TGCCCACCCTCTAAAACCCAAACAAAAACAACAAAGTCAAGAAAACCCA
TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC
ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG
GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT
GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC
AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT
CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC
TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA
CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT
TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT
TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG
CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG
ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC
AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG
CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA
AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG
CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT
ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG
AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC
CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG
GCATAATTACTAGCTCATGGACTGACAAGGTCTACACTATTTCGAATCTCA
CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC
TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC
AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT
CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC
CCTCTCAGCAGCCAGGTTTTGCGTTCCATAATGTCACGTACCCCCAGTCAT
ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC
TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT
TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG
AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC
TGACTGAACATTCTTGCTGGGCCAAGTCTTAATCATCTCTAAATCCCTCTG
GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA
AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT
TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA
CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT
CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA
TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG
CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT
TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC
ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA
CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC
AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT
ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG
AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG
TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC
AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT
ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG
ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC
CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA
AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT
CCTCTGTGGCTCACTGACTTCAGAAGCCAACC~'CCGTTGTTCAAGTCACTT
GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG
GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT
GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT
TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT
TGGCTTTTTTCTTGCCATTACCCTACTTGGCCAAGTGCAAATGGGATTTCA
ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA
CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC
CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC
CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC
TCCATGGGCTCTTCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCT
GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT
CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA
TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT
TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA
GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT
GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC
TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT
TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA
CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA
CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC
CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG
CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC
AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG
AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA
GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA
TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT
GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA
GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC
AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC
CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC
AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT
TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA
CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG
GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG
AAGGGGAAGGGAGGACAGAAGAGGGGAGGGAGCGGGGAGATTATCTAC
CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT
GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC
TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT
TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT
GACTTTCCCTTTGCAATGACAGGAAGATAGTATACTGCATGGGACCACCA
CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC
CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA
CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA
GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA
ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG
AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG
GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC
GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA
ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA
CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA
GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA
GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC
ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG
TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC
ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG
GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC
TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT
GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT
TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC
TCCTCCTCAGTCTTGGCCGGTCTGGGAAGAACTTGGTTTAGATGCTGGGA
ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC
CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA
CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC
TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG
GGGCCACAAATCTGCTCTGCCACTTCACTGCTGGGTGAAAGCTGTTGGCTT
GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC
TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC
CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT
CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA
AGAGTGAACAGCTGAGGAGCTCTGTGACTCTGAGAGAGAGAGAAATCAA
TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG
GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG
CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT
CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT
CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC
CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG
GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC
CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC
TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG
GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG
GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG
GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC
CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA
CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC
CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT
GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA
GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC
TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG
GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG
GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA
GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC
CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG
AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG
CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG
CTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATCAGCTGCTT
GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG
GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA
ACCGCGGCCGATCTGAATTCAGATCGGCCGCGGGATCATTCCTGGACTCA
GATTGTTCTGAAGAAGCCCAGTTCTGGGTGGCATCAAGTGCTTGCTAGAT
GGGGGGCTTGCCTTGATCCGGCTACACTTGGAGGTGACTTGTTCTTGGAC
GGCTACATACAGAAAGAGAGAAGTGGGGATGAGTTCCAAAGGCATCCTC
GACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACGGACTTC
TCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTCC
TCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGG
GAAGCTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAGCAGATCACACT
GGACTGAAATGCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTG
GAGGTCAGGGTCCATGTCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCT
GGCACTGCCCACCCTTTGACAGGTGGTGCATATTTTGTAGAAGGAAGGAA
GGGGCCAGGTGGGGTGGGCTGGGCTGGTGGCGGGAGCTAGCTCAGCCTCT
TAGATTCTCTACCCGATGGATGTGACCTGGGACAGCAAGTGAGTGTGGTG
AGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGC
CTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGAACGGTA
ATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTCC
CTGAGTTCTGAAGAGCCTGGGGATGGAGAGACACGGAGCAGAAGATGGA
AGGTAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTC
ATGAGAGTCCACTCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACT
TTTTTGGGGTGGACACTGAGTCATGCAGCAGTCTTATGGTTTACCCAAGGT
CAGGTAGGGGAGACAGTGCAGTCAGAGCACAAGCCCAGTGTGTCTGACC
CACCCAAGAATCCATGCTCGTATCTACAAAAATGATTTTTTCTCTTGTAAT
GGTGCCTAGGTTCTTTTATTATCATGGCATGTGTATGTTTTTCAACTAGGTT
ACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCACCAGCCTTCCTGA
AACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGGCACTCCA
GCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGTC
TTCCATAAGACCAGAAGAGCAATGGTGTAGAGGTGTCATGGGCTACAATA
AAGATGCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCTGGCCCTCAGA
CAAATGCTGAGTGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCC
ACTGGTGCGTAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAAC
CTCCACGGAGTGGGAACTACATCAGGCATTTTGCTAACTGCTGTATCCTA
GGCCAATAAATGTTGATCACATTTATAGCTGCCATGGTAGGGTGGGGACC
CCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCCTGACACAAACTTTCTGA
AGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCC
AGGAACCCATTAGAAATGGAAGGTCACTGCAGCAGTATGTGAATGTGCGT
GTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGT
GATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGC
TTTAAGGGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAAGTGGTGATTT
TGGAGTCATTTAGACCAGGCCTTCAATTGGGATCCTGCTCTTAGAGTTGGA
TGAATTATTTAACTGATTTTCAGATCTCCTCTTTCTCAATGCTTTCAGAAGC
ACAGTAACTGCTTACTCTGAAATGAATTCTCACCCCACTTCCACATATGCA
CCCCTTGCCCACCCCTTTGGGAACACTGGCCTTAACTGCTTACCTTCAAAT
GGACTCATCTGTTGGGAGATATATGCATTCTGCCGTTCAGGGGTCATTGCC
ATAAGACCTGATCTCTGTTCCTCTTGCTAAACAGAAGATGAAAAAGACAA
ATTAGATTACAGCTACCAATTAATAATTAGCCTTAGGATCGCTGCGTGGG
GACCTAGGACTTGGCTTTGGTGCAGCAGAAAGCATGAATAAACACACCAG
CATACACTCGCATGCATGCCCCACCCTCTCGAGCAAAATTCCACAGGTAT
AAATAAAGTAAGATTCTGCACCTGGGTTAAAAACACAACTGCAACAGCAT
AGAATGGGGCAGGAGAGACAGAACTTAATAGCAAGAGCACACAGAAAAA
AGTTTTAGGCATTTTGGATGTCCATCTGCTCAGGATGGGTCAGCAGTGAG
ATGCGGTCACCAAAAGAACAAATGTAACATTAGGCTGCATTAATAGAAGC
AGAGTATGTAGAAGGAGGGAGGTGACAGTCCTATGCTAACTCTGCCTTGG
CCAGACTATACCCACAGGAGTCTGGGCATGCCAGTCTCAGGGAGACCCAG
ACAGACTGGCTGCATTCAGAGGATGGTAAGTAATGAGAGTGGGGATTGG
ACTTCAAACTACCCAGACAAAGAATGGCTGAGCAAGCCAAGGATGCTGT
GGCTGGGGCAGAGCAGACTGTGGGCTATGTAGTGGTGGATACCTAGCCTC
TGCAGGGCTGTCATAGGGAAAGGACATTGAGAAGAGGACTGAGGCTTGTT
CCTGGTGGTCCTGGCATGAACGGCCAGATGATCACATGGTCAGGTGGACA
CAGTCTCCAACACTGGGAGTAGCCAAACACTTACTGCCAACCTCCCGCCC
TTCTCCTGACTAGTTGCAGCATAGGCAATTGGGAGGAGCTTCCTGTCTCCA
TCTGAAAGCTGGCTGGGTGGGCAGGGGGAGGAGCGAGCCAAGTTTCAAG
GCCGCAGTTTCAGCACTCAGTCTGGGATCGGCTCAAGGAGCAAAGGGGA
AGAACATAGCCAGGAGGGAATAACATGAAGGCCCCCAGACCCAGAAAAG
GCATGACTTGCTCTGAGACCCTCAGCCGGTTGGTGTCAGGTTGTGACTCG
GATCCAGGTCTGACTCCCAGTCCAGTGCTTGAAGCCTCACCCCACACAGT
GAGGGGAGCCCGGCCATCTCTGCTCAACTGCTGCCATCTCTCTCCCCTTCT
CAACCACCAAGGCAGCTCTGTCTGGGAGCACAAGCTCCAAGTCCACTTTC
TGGTCTGTGTCCCCCCCAAGATGCCAGAGGACTTGCCTCTACAACACGGG
CTGCCCGTGCAGTGCCTGCTTTTCCAGCAAAGGGCTTCTGGGAACCCTTCT
CTGCACTCAGTGGGGCTGGTGGGAGTGGGGCGGGGTAGCGACCCAGTGCT
TGGGACTGTGCCCAGCTCTCAGGCCTGGCAGCAGTTCCTGGCCTTGGTTCC
TGCCAAGGCAGAGAGGACAAACACATGGCACCGGGAAGACTACACCAGA
AGCGATTCCACCAGACTGGGGTTTGCTTTTCTATCCCGCCCTTAGCCTGCT
TCCTGTCCTGGTCCCTGCCTCCCCCTCCACTGGAGCTGCCGTGTGGGCAGT
GAGGGGCTGTTTCTCAGCTGCCCTATGGAGCTGCCCTCTCCCTGCCAAAGC
ATTGGCAAGGCGGCAAGGGGTGGGGGTGGGGATGGGGGGTGGGATCTGC
CTTCTCAAGCTCTCATTATACTGAGCACGTCTCACCCATTATTTTATGTCAT
CTAGCAACACCCCATGTGGACACTGAGGAGCATGGGGGTCACATGACCAC
TGCCCAAGGCCACACCATCCGGATCTGCCTGAGATGGTCAGGGTTGGCAG
CCATTTCTGAAGGCAGTCCTTTCGCTTTGGCTCTTCTTGTACCAGTCTCAG
GACATCAGGGCAGAAGATCTACAGTCCCCAGCTTACTGATGTGACAGCAG
AGGCTCAGAGAGGTTAAATGACTTGCCCAAGGTGACACGGCTAAGAAGT
ACAGTATCTCCTAACTGCAGACCAGGTGCTTCTGCTGCTTCTGGGGACAG
ATTCCTGCGTGGCTGGCTAGGTCTAAACGGTCCTTAACTCCATCCCCACCG
GTTGCTGCATTAGTTTCATCAAATAACACAGTTGTACAGAGGTAGGGGTT
CAGGGGCAGGGGCAGATGGAGGCTGGAGAGTGTGACTAAGGAAACAGCA
GGGGAAGTGCGGTAAAGTCCGAAGGGAGGGACGGAAAGAGAAAGCCAA
GCCCAGGGGCGTGCCAGACAAAAGGAAAGGCCACGCCGGGGCAGGGCAG
GCTTCAGCGGGTGCTGGGGCGTCTTCATCCCGGGAAGCACACATTCCAGA
GGACCCCGGAGTCTAATGGAAAAGCTGGCCAGCCTATCACTATGGAAACT
GCCAAGGCCACACAGCGCTACGCGTATTTAAATGTGCTGGAGTGTTGAGA
TACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGTGTTGGGATGC
CTAGGTGCTGGGGTGCCGAGATGCTGGAGTACTAGTGTGCTGGGATGCTG
AAGTGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGG
CTGCTGGAGTGTTGGGGTGCTGGGATGCTGGAGTGCTGAGATGCTTGGAC
AATGGGGTGCTGGAATACTATGGTGCTGGGGTGCTGGGGTATTGAGATGC
TAGGGTACTGGGATGCTGAAGTGCTGAGATCCTGGAGTGCTGGGCTGCTG
GGCCACAGGCTCTTGAATCCATTCGTCTGCCCAGGGGAAGAAACCAGAAG
ATAAAGAGCTAATGAAGGAGCTTTGGTTGAGAGGGAGGAAGTAATGGAA
GGAGCAACATCTTGTGGAGGAGCAGGAGAGAATGGACCTCAGGTTGGGA
GAGAGGGCCAGGCTACAGGCCAGAGAGGCAGAAGGATTCCAGCAGAGTG
TGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTCCA
GTACTGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCT
TTATGACCAGGAGGAATTTGGAACTGTGACTAGAGTACTTAGGGGGAAGG
AGGCAAGACTGGAGCCAGATTGCTCTGGGTTGAGGGGTGAGTGGGAGGT
GAAGCAGGGCACTGTCACTCCTTTGAAGGGTGGCAGAGAGCTGGAATTGG
TGCTGGATGGGCTGTGGGGTGACAGGGTCATGTGGAAAGCCCCTGGGGGG
CACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGAGGACAGCTAAGGGA
AAGCGGAATGGCAGAACACGCACTGCCAGGAGGAATGAGGATAGGGTCA
GGAGTGCCAGGGGCAGTGAGGCCAGCCTGGGGTCAGGTGGCAGGACGTG
TCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCTTGAGGTT
CCCTGTGTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGA
ACCAGCTCTGCGAGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTG
GCAGAGCTGAGTGGGGACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGT
GGCCCCTCCGGGAGGGCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCA
GTGGCTGGGCAGGTGGGTCCAACACAGAGCCAGAGAAGGGTGAATGGGC
CTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGAC
AGCACAGCTGAGCCCAAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGCAG
CCTGCCTGGGGCCTGGCATGGATGGGACACTTGAGGCAGAGGGACAATA
GTGGGCGCTGCAGTGAGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCT
GCCTGAGACGGGCAGGGAGAACAGAGACAAAGTTGGTGACAGGGAATGA
AAGCTGACTGAAGGACTTTACCCAGACCTATGAGGATATCTCTCTCAGCA
GGAAGCAGGAGGGGACTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGG
GAAAATGACTCTTTCTCCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTG
CGGAGGTCTGCTTCCACCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCC
ATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCG
GGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAG
CCACTGATCAAATTGGAACATTTTGTACCTTAGGGATGAGGATATCAACT
CTCCCAGCCACTTAGAGGGATAAATGTGATGATGCATTCAATTGTGACTA
CATCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGA
GGCGTGCATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCAT
CCATGAAACCTGGGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAG
GGTCTGGTCAAAGAAGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGAC
GGTGGTGCAGAGCGTCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCT
CCCGGGGCAGTTGTCTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTAT
AAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAA
AACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAA
AGAACCAAAGCCACTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAA
TACTCCCTTGAAAAATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTAT
TACAGAACGAAGAAAAAACACACTGGCTTGAAACAGACGCCAGATTTCA
AATGTAGAGGTGAAATACGAGGTGGCAATTAAAATGTGATTACAGAAAG
TCTGGACACTGAGAAAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAA
CAGACACTTAAATATACATGACGATAATTGCAGATAGAAACCATCAAAGA
CAAACCCCAAATCAACTAATAATGTTTACAGATGTTCCCCCCCAAACCAC
AGAGCCTTACATCAAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCT
CGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTG
AATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGA
CAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAAC
TCTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAAC
AGCTTCTGCTGTAAAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGT
TCGCAAAAGAGATTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACA
TAGAAATAAATGGCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACA
ACAACTCTTGGTCATATCCACATTGCTCATTGCATTAAAACCATAAGCGAC
TCAGCCACCTAGCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGC
ATATTTGTAACATTGTATAATAAACACAAAACAATGCATAGTAAACACAA
CTCTACTGAAACAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAG
TATAAATACTTCTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCA
AATATGTACACGTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGT
AGAGAGAGGGATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGG
AGACAACTGCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCAT
GGGGGTTTCTATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTG
GGTGACTTGCCCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCT
GTTCCAAAATGTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTG
GGCTCACACAGGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTG
CAGTCAGGTGACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAA
AAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGAC
CTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGC
AGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGAC
TAAAGCTGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGC
ACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCT
GAAGCTGGGGTTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCA
CTTGTGCCTCCCAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGT
GAGTGCTGCTGAACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCCCGAA
ACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAA
CGCAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCA
AGGACTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTC
TGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTC
TGCCCAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCT
GGAGGTGAAAATTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCA
TGCCCGGCGCGCGCGTCGACGTGAGCAAGTGGAATGGAGTGGCAGGGAA
TGAGTGAGGGAGACAACAGCTCAGTAGGACCTTGTGGGCTACTGGAAAG
ACTTTGGCTTTGACTCTCAGTGAGATAAGGAGGTATTTGAAGGAGTTTTAG
CAGAGAAGGAACAATTCTGTTTATTTTTAGGAGCTCTTTATATATTTAGGA
AATTACTTCTTTGTTAGTCATATATTTTGGAATTTGTTGTTCCCCGTTTGTC
ATTTCTGTTTCTACATTTTGCTTATAATATTTGTCTATGTAAACCTTAATAT
GTACTTGGTTAAATTGATAAATTCTTTGCATAATTAATAGCTAGCTATTGT
TGGCCTACCTATAATTAATTTTTGCTTTATCTAGTAATAGGCCTGAATTTTC
ATTTGGGATCCAGCCCTCTCCCTGACACTATGCAGGGCCTGGGAGACTAA
AGCCTGCTGGAAATAACTCCAGCTTGGCCCTGCCAAACATTACAACACCT
AGCACCTGCTCTCCCCACCATAGTGACTGATTTGGGCAGGAATGAGACCA
CAGCCACTCCAAGAGCCCAGCTGTTGGCTACCAGTAACATCTTCTCCTCTA
AACAGGAGATCCAGCTATATTGAAGGCTTCCAAAATTACCCTATGTCTAC
CTGTCACAGGAATGTTCAGAGGACTAAGCCTCTGCCTTTCTCCACCCCATA
CTTACTTCATATGGAACTATGAATGGGCAACTTCACAATTTATTATTTACG
TCACCTAGATGCAAGTCAGAGGGAGAAGCAACAAATCAGCAGTACCTAA
GAAGTTCTCAAGTCCTCAAGGTGAACTTTACCTATATATACAAAATATATT
TGCCTCTTTCCTTTAACCCTTTTTCTTACTCCCACCACTTTCCACCCAATGT
GTAATAATATTAGTCACTTCATAGAGTTCTTATGAGAAGTTAAATGAGGA
AATCATGTAAACCACTGAGGGCTGGCAACAAGTTCTTAATAAGTGCTATT
GAGTTATGAGTATTATTATCTCCCTTGGGCTGGATTCACAGTGTCACCTCT
TCTGGGACCATAGTAAGCCAATGGTTCCAGCTCCTTCTATTATGTCATTTT
AATTTGGGGAATAGCTTAACATATTAGATGATCCTTTACATGCCTGAGCG
GTCTTTCTTTGATGGTATTTCTATGATCTTATGAAGAGCTAGATGTTGCCG
CTCCATTTTATCTTGCTGGTGGCAGCTGGTGTTTAGCCAACTTGTCTTTTTT
TTTCTGTCTTTATATTTTTGGTTTTGTTTTTTGGCAGAAGCCTGTGTTTTTCA
GATTACTTTTTGCTTGTGTCTACTTTTTCCCAAAACAATATTACATATTAAA
CTTATTGTATATTTAAGTTTATTTTATGCATCAAAAGTCGCATTGAATTTCT
GTGCTATTGTGGGGCAAATTGACATCAATGTAATTTAACTCATTTTATCCC
AAATTAGGCTATGTCTTTCCATATACTCAAATCTTCTTTTATGTCCCTCAAC
AGTTTCCTTCATATAAATATTGCATATTTCTAATTAAGTTTATTTTTCTAAT
CTCCATTTTGGATGTCTTAAATATAGTCTTTTCTCCCTTTGCTCTTTATCAA
AGATCTGCAAACTATGGCCTGTTTTT'GTAAATAAAGTTTTGTAATAAAGTT
TAAATGGCCACTCCCATTTGTTTACATGTTGAGTATGGCTGCTTTCAGACT
GAAATGGCGAATGAAGTGGTCATAACAAGACCATATGGCCTGGAAAGCA
AATATATTTATTATCTGGCAATTTACAAAAAAAGTTTGCCAACCTCTGCTT
TATATTTTTGATCTAGTTGTTATTTGAAAATAAAGCTATTGATATTTGTATA
TTAATTTTTTACCCAGCTACCTTATTGTTTTTATTACTTAATAGTTTACAGT
ATTTCTTTTAGTTTTTCCAGGTGTAACATCTGTAAATATTAATAAATCTATT
AGCTAATATTTCATTTAGCATTTTTATATCTATACTCACATGAGAAGTTTT
CTTTAATTTTCTTTTGTGTTTATCAACCTGTGTTCACTCAGGAAAATGAAG
CCACTTAAGTATACCAGGAGTGGAGGGTTTCATATAGAAATGAGGGCTTA
TTTGAATTTTGGGAAAGCTGATGAAACAGGTCAGCAGAGTTACTGCAGAA
GACCAGGAAAGTTACATCTGGAAGGTTAGGGAAGCAGACACTAAAGACT
TAAGCCTGAAGCATAGAAGTAAGGATCCGTCAATACTTATTGAGAAACTT
CTGGAATTCTCAAGAACCTCTGAGAAATCTCCCATTCTGTGGCAATAAAG
TGGTGGTACTCAAGCCATCACCAACATGGCTCATATGTCTCATTGCACTAT
GTCCAAAGACAAATGGCCTCTGCTTCTCTTCTGTCTCCTAAATCTCTTAAG
ATTTCCTCTTATTTGTTTAAGTCTAACCCAGAAGACTTACATGGCAGGTAA
TTCTGGGAAATGTATTCCCTTGTTTCTCCAAAGGGGTGGGGATAATACCA
AGTTGACAACAGAAAATCTAGCACCCTTTTTAAGGTTTTGGCATCAGTGA
TTTCTTGGTGCTCTAAATAGTTCTTATGATTATTCCATGGGTGCTTAAAAA
GAATATGTATTCTCTGATTTAAGAATATGTAATTGATTTAACTATTGATTA
GATATTAATAAAATAGATTTTTAAATTACCTTATTAATTTGGTTAAGTCCT
CCATGACTACTTATTTTTTGACTATTTGTTATGGAAAGGTGTAGCTAAATA
AATATAGAACTACTCTATATGTGTTTCTAGTTCTCTATGTATTCTCTACATT
TTGCTTTATAATTGTTTTCTTTCTTAGAGACAGGGGTCTTGCTGTGTTGCCC
AGGCCAGAGTGCAGTGGCATGATCATAACTCACTGTAGCCTTAAACTCCT
GGGCTCAAGCAATCCTCCTGCCTCAGCTTCTCAAGTAGATGGGACTACAG
AAGTGCATCACCACACCCTGCTAATTTTTTTCTTATTTTTATTTTTAGTGAT
GGAGTCATGCTATGATGCTCAGAGTGGTCTTGAGCTCCTGGCCTCAAGCA
ATCCTCCCACCTCAGCCCCCTGAGTAGCTGGGATTATAGGTGAGAGGCAC
CATGCCTGGATTTGCTTTATAAATTTTGATATGTTACTTGGTGCATAGATA
TTCATTGCTGTTAGACCTTCATTATGTGTTGTACCCTGCCCTTCTTTGGTTC
ACTTTTCCTTGAATTCAGTCTTGTCTGATATTAATTTTCAGCTGTCTGCTTT
CTTCTTATTTCCATTTTTCTAATATGCTTTTGCCTACTCATTTGCTTTCAATT
TTTTCTGAGTCATTTCATTCTAGATGAGTATATTACAGTCAGTACTTAATT
GGGTTTTTCTTTGTCACTGAATATGAGTGTCTCTTTTCCTGTGTATTGATAG
GGTAGATATGTGTAGTATGAGTGTTACAATTTTTTAAAAAACTTTGATTTC
TCTCCTTTGTTTTATTTGGGGTCTCCGGTTATTTTTTAAGGTGTATAGTGTC
ATTTTATTTTTAATGTTTGCCTCTGTAAAAATAATTTTATAGGCTGTACTTA
ATCTTCTATTTATTTAGCTAGTTTATATTGACTCTTCAATAACAGTAGTAA
ATGTCCCTACTTCTAACTCCCCTTGCCCTCCACCTTTTAATTTTGTGGGGTA
TACTACTGGCCTTAGTTTTTCTAGTGGTTAATTTTATACATTTAAATAGGTT
TATACCTTTTAAACTTCAATGGCCACCTTTAAAAATATTTTGACCCCACCC
ACTATAAAAAATAAACATGCCATCTTACACGCACAATCTCACTTTCTCTCC
CCTTACATTCCCTAATTTTTACTGGGTATTTTAATATTTCTTCATCATCGTG
GTTGATTACATTTACATGTATATTGCAGTGGTAGTTCCCATAATTGTTATA
ACCTTTAACCATAGTTCTACATTTATTTTTGCCACCGTTTTTCCCATTCACC
TTTTGGTTCAACTTTATCTTCTAGTAGTGTCTTCAAAAAAGTGTTCTTGGA
AACTACAGCCCATGAAACATTATTGCATATTTGAAAATATTTATCTGTCAC
CATTACACCTGAATGACAATTTAGCTAGCTCAAATTTACTGGCTCACATCA
TCTTTTTTTTGGTAATTTTTTAAGTACTTCTCTACTGTCATGTAATGTTGTA
TGTAGGATAGCCTAAAGCCAACTTGTGTTTTTTTATTTTGCTGTTATAGTA
GTTTGATCATTTTTGCCTAGATGCCCCATAGGATTATTTTTCTTTGTCTTTA
TTTCAGGAAACCTTACTAGACTATGGTTCAGTGCTGACTAGACATTTTTTT
CCTTAAGAAACACCATGCCCTTTAGATCCTTAGATTCAAATCTTTTTTAAT
ATCAGTAAAGTTTTTAAATTTACATATTTGGATATTTTATACTTCTCTAGTT
TTTCTTCTTAAAGAAGAAAACATTAATTCTGTCTTTGCCTGTCTTCCATATC
TACAATTTTCTTCTAATCACTTTTTATAATTTCCATTTCATTTTGCTCAATT
GCCTCAAAGCTGCTATCTATTTTTCTGTTGTCAACAATCTGTACTCTTACAT
CAGCTACTATGATAATATTGTGTCACCATTTACTACTATTTACAAGCTAAC
CATAATTTCTCTGATGATTTTAGTTTTCTCTTCTACTTTGTGTCCTAGATAC
AGGCAGCTCAATTTTAATTTTTGTTGCTTTTCTTGATTCCTTATACCTCTGC
ATTAAGCTCTTAATTCACAAATTACTTCCATTGTTAAGTTATTGAGTTCTT
AAACAAATATTTGGTCATGATTTTCATCTACTCCACATCAAAATGTTCATA
CTGTGTAGGCTGTTTGCCTTTTTTGTCCTGCTATTTTCCACCATTTTGTGAC
ATTTTTGTATGGATCTCCTTGTTCGTTTTTTATTATTAGTCATCCTTGAGTG
AGCAGAAATCTTCCTAGAGCAGGTATTTGAAAGAAGTAGTGTGGAAGAG
GGGCTGGGGCTGTGTTCTGCGTTAGTAGAAAATCCCCTAATTCTTAGAAA
AATCCTTTATGATTCATGGTCTTGTGGGAGAAGGTTGTGAAGCGGGTTTTG
TGAATGTGCTTCATGTGCAATAGAGAGTGACTGAGAGGTGGTTTAACCTT
TCTTCTTAGCTCACAAAGAATGGCTGCTACAGAGCTGCTTAACACCCACA
GTTTTTCTTCTACATGCTGCACATTTACAAAGATGGAGTATCCGTGAGTTT
TCTCACTCATTCCTACCTCCCCTGCCATTTTAAAGTATCCAAGGAAGTTGG
GGAGATTCCCTCAGCTAAACAGCCACCCAGATCTTATTGTTCCAAACAAA
AAATTATTAGATTTGCCCTCTGAGCAGGTCACCTACTTTGGAGAATTTTGT
GCTCTGCCAGCTCTCCTCAAGATTTGGAAAAGCAGTTATTTTCTATTGGAG
CCCCCATCACTCCAGTCCAAAGTTCACTGCATGTGACATCCCATCTTATTT
GTCTGGTTCCTGATTATAGGAGTTTCCCAGTTTCAACAAAAATACTGTCTT
TGTTTTTGTTTCTCATTCATTTTTCCCCAATTTTGTTTCAAAGCGAAAAGTA
AAAACATTCTTACTCTAACATCTTAAAATTAATGTCTGGGAGGAACCTAA
ACTGCTCATTAATATATGCCATGCTATATGTGATATGGCAGAGACTGCTAC
CTGTCCACCAGAATCTATTTATTTTTCTTGGATGCACAATAAGACTACATT
TCCTAGTCTCCCTTGCAATTACATGTGACCATGTCATTAAGATTTAGTGGA
TGAAATTTAAGCAGAAGCAAGGTGTGTAAATTCTAGGGCTGGCCCATTAC
AAACTTTAACATGGGCACCTGTGTGTTTTTCCTTTTCTGCTTAATTGGGTTG
GAGATAATCACAGTGATGCTGAAAGCCATGTGCTAAAGAAGGCAGAGCC
TCCTTCTGCTTGGGCTCCTGAATGACTGTGTGGAACAGAGGCCCTTGCCAC
TCTTGGGCCATTCTGGATTATTACATGCACAAAGAGTAAAGTTCTTTTCTG
TTAAGCCATTAGATGTATGGGTTTATTTGCTGTTACAGCCTAGTCGAACCC
AAATGATGTAGTGAGGGAGGCAGGACAAGGAAGAAACCTATTCAGTATG
GAGTTATCCTTAGGGACAAGTTACAAAAATTATAAGGATCAGACACCACT
GCTGGTTGATCCATTATAGACCAGAATAAGCAGGTGGTATGATTCCAGAA
GGAGATGCCATGGTTGTGTGAGAGAGAGATTAACCATTCCTATAAACAGA
AGGGTCAAAGAAAGTAGCCAGTCACCAGAATGCTTATATTAAGGAGGATT
GTGACTACCTGTATATAAAGTATAGGAAGAAAATGGGGCAGGGCAATTA
AGATGAATTGCAGACACAACTATTTGTGCATATTAATTTGAATTTAAATTT
AAACTTAATTTAAGGATAATTTAAAGGAAAATCTTCCCTTACCTATTTCAT
GCTGAAGGGGTCTTTCAAGTTGTCAAACTCTCTCTATATAAATAAGACTGC
ATAATCCAGACCCTCCGGAATCCAGAGATGAATTGCATGTGGTAGCTGAT
GTGTGGGAAGCCCACAGAGCAGCACTTCTGGTCATTGCCATCCAGCTAGC
TGGTGCTCTCAGAAGAATGAGATGAAGAGGCATGACTTTCAAGCTTCCTC
TGCCATACAAGGTGAGTTTGGGTTCCAGGCTTCTTCCTAGCCCCTGTTTCT
GCTTCCTCTGATTTAATACCCAGTCACTGAGTACCTACTAAACACTCGGCA
TTAAACTGAAAAGAACAGGGAAGTCAAAAACCTTTTACCAATGCCTGATG
GGATAAAGACATGACCCATCCCTCAAAGAGCTTGTGGTCTTCCCATGGCA
ATGCCCTTCCTCCCTAGGCCAGATGGACTGTGAAGAACAGCAGTCTCAAG
TGGATTTATTGGGAAGCTCATGAAGACTCAGCTCAATGGGCCATGTTTGC
AGGGGCCCCTTACAAGGCTCTGCACTTGATTTTGTAATTTTGAATTATTTT
TCTCAAAAAGGCCTCCCAAATTATGTAAGCTTCAGGCCTTTAAAACTTGG
ATCTGTTCCCTTGAAGCCACATAATCTGATCAGAAATAATTTTCAGGGCTC
ATTGGGTGCTGGAGAATTCATGGTTAGATATTTAAATAAAAATGCAGAGA
ATCTATAGGACTTTCCAACTGAGACCAAGGCTTTTTTTTTCTGGTAGCTTG
ACTGCCAGGAAACAGAAAGCGGGGCATCTCCACCTGCTGGCAGAAGACC
AAAACTACACCATATTGCATTGACCGCAGACATCAGGCTTTGTTTCCTTTT
AAGTGAATAGGTGAAATCAATCAAGCAAACTGGAATTAGGAAACAGAAG
GACCACTCACAACCGTACCTCCACAGCAAAGCTAACACTTTCATTTTGAA
TAACCCCTTTCCAGCCCCTGTTTACATCAGTCACATTTTTTGCATGGACAT
AATAGTGTTTCAGGCAGTCCTGCTCTACAGAAGGCATTTGTTTCGCTGACC
ATAAGCATCTTATGTGCTTCTACATTGTCTTTATAGCCAGCTGTCTGAAGT
TCTACATCATCCGTCAGTGATGGGTCCTGAGTAAACTCAGCAGTCTCCTCT
AAGTAACTTGTATATATCCCTTTTATAAAGAATGCTGCAGTGGCCACCCTT
AAGTATAGAACTTTGTCTTTCCCAGGCATTACTTACAGGAAACTGGGGAC
CAAATGCCACTCCATAGCATAGCTTAGGAGAAGTGAGATTCCAAAGGACA
GAGTGGGCTGGCACATATGTAGTAGAAGACTGGACAAATGAGAGTGAAT
GAGCGAGTCTGTTGTGAGTGGATGACACGCAATGCAACCCCAAGGGCCG
GCTCCAATTTGCACAGTCCCTAAAACATCCCAGGGATCCAGTCTTATCGC
AGTCTCATCAACATTGACAATAATCATTTAGAGAATTTTCACCATGAGACT
TTTGACACTCTGCTCTGGGATTTTGGAGTTCCAACTGCTTTAACAATTTAT
ACAGTCACTCAAATGTAGAATCCAGTGAATTATTAATGACAATTTTGCTCC
GCATATTGAAAGGGCATTCTATATTAATCAATTATAAATCATGATAAATTG
CTTTCTAAAAATTATACACTTGAAACCCAGAAATGTTCAAGCCCCTCACC
AGTTTACATACATCCTTAACCGTGTCTTGTTAACTGGGTGACCAGGAAAC
AGAGAAAGGAGGATGTACTTAATGAATAACTGGCCAGAAGTCGGAAGAC
CTAGATTTTCTTCCCATCCCTTCCATAGATTTGCTCTGTGACCCTAGACAA
TTCATATGCCCCCTCTGTTACCATACTTTTAAGCTGAAGGGGTTATAGTGC
ATACTTTCTGAGGTCCTTCTAACACTGGAGATCTATGAGTTCATAAGATCA
CATAGAGTCTTGGACTTTTCAGAGATCACTAGGCATAAGAAAGCCCTCGA
GGACATCCTCTTCCTAGATCAACCACTCCAAGCTAAGACCTTAATGGGTCT
ACACTGTCATCCATTTATATAGGAGAGAGGATGGTACAAGCTAAGTGAAA
GTCTGTTGTCAACTCAGGAACTAGGGTACAGTTCCAGCAATCAATCTCCA
TGGAGGGTTAGCAGAGGTCTGGCCTAGCCAATTAGGGTTTGGCACTGCCA
TATGCTACTAGAAAACACATGCTTTCATGCCAGGCAACAGGGTGGGAAGT
GCAATGCCATTAAGGACCATACCTTAGCCTACTTAAACTAGCCTGGGATT
GAGTTACTGAGCTCAAAATACAATTCAGCACACTTTCCTGCCCACTGCAA
GTCAGGCCAGCCCCTGGAGAACTCCAGGGACAAGGCAAGTCTGGAGTTTG
TCCCCAAGGAACTCCCAGTCTAGTGGAGGAGATGGGCCCATAGCAGAGTT
ACAAATGTCCAAAATAAGAGCACAAATTCCTGGGTTCTTAAAGAGAGTTC
AAGCACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAT
TAATTTAGATAAGGAAGGGAGGTAGGGAGAGATGGTGCCAGCAATGGCT
TCCTGAGGAAATGTCACTTGTGATGGGTCTTTTAGGTGAGTTGGACATTGA
GAGAAAGTAATCAAAAGGGCACAAGATAGGGTAGCTGTCAGGTAAACAA
AACAAAACTGTATGCACAAAGGTCTAGAGGCAAGAAAGCATGGGTGTGC
TTGCATAGCAATGAAGAATGTGGTTTGGCCAAAGCATAGGATTCACAGAA
GGACCTCCCCAGGCCATCAGTAGTCTCTCCTTCGCAGGAGTCCACTGTGG
CTGGACGAGGAACAGATCAAAACAGGTCTTGTTTGAGGCTTGGCCCAACA
ACAGGGCCAGCTGTTGACCAGGGCTTCCTTCACCCTTAGCTTTCTCTCCCC
CTAGTGTAGTCAGAGACTCCAAAGGCAGTCCCAGAGTGTTGTGCCAAAAC
AAATGCACATCAGGCTATTGTCATCAGGAGCCCTTGTCTTGTTCTGTTTGT
TTTAAGAAGACATAAGTGAAATAACCAGGTTTCCGAAGAGAGGGCAGAA
TTGGGTTTGTCCCCAGGGAAAATGAGGTTTCTCCACCATGGCTGTTCTCCC
CATGCAGCAGCGGCCAGGGGTCCCCAGCAGCTCTTTGCTCAGTGCCCCTG
ATGCTAGGCCTGATGCTTCCTATGTGGTCAGCTCAGATGTGCAACCCTTCT
CCATTTTTATTATTTGAGCAATATAATCATTATTGCTTTTGTTACCCAAAAT
CTTATAATCACCCATTTAAGAGTAATGTAGGAATCATTTTTCTCTTTTATTT
GATGAACTAATCTTTCTGGTTGGCTCTTGAGGTGGGAGGGGTGGGCAATG
GGGACCAGCCTGCTTCATCTCACTCTTTCCTAGGCCTGCTCCTTAGGCACG
TGCCCCACAAATCCCACCTCCGCAGGTTGGAAAAGGGGCCCACAGAGGAT
CTCAGGTATCTGAGGAAGGGCCAGAGGCTATTTGGCCTGACATCCTGGAA
CATCACCAGCCAGGAGCCATAACTCCTTTAGGCTAGCCTGCCTGCAACTG
GATCAGGTATAGCAAAAACGATTGATCTCACCTTCTAATTTTCAATGAGT
GGGAGTGGCTGCCTTGAGTGCTGTGTTGGGAAGGAGTCTGAGGCAGCATC
TGGACCCTGTGTAAATATGCGTTTCCTGTTATTGATGAAGCTTTCAAGACA
AGATTTTAGAAAGCTTTGGCTCAAGTTGTAGGTTGTGGGAGGAAGGAATT
AGTAAGAGCCATCATAAACTCAACATCTATTGTGTTCCAGGAGCTGTGAC
CACTCCCTTCTTTTTAGTCGGGAAGGTCAGGATCTCACTATGTCACCTAGG
CTAGAGTGCAATGGTGTGATTATTGCTCATTGTAGTCTTGAATTTGAATTC
CTGGGCTCAAGTAATCCTCCTGCCTCAGCCTTCTGAGTAGGTGGGACTACT
GGTGTGTGCCACCACACTCGGCTTACTCCTCCCTTTGTATCCTGCTGCATC
GTTAGTGCATGATACCCACCACCCCCTCAGAGGCCCAGCCTAGTGCTGTC
TCTCAGAAAAACACCCTTTGAGTCAACAGAGACAAACTTAGGGGGTGCCT
GACTCCAAAGTCCAGGCCTTCCTCCTTTGAGGAACAGCATGGCTTCCCCA
GGGAATATCATTCCATGGAGACAAGGCCTGCAGACAGATCTCATTTTCAT
CTGTTCTTTTACTAGCCCTTGGAGGATTTGCTCCAAAAACCTGAGGCACTG
ATGTTTACTCCAGCTTTACTTCCCCCATACCTGGTGAACAACTCTGGCCCA
GCTTCCACACACCAAGGCCCATGGCTCTAATAGAGCCAGAGAGGCAGTCT
CAGCTAAGAAGAGCTGTATCCTCCAGGGACCAGCAGGAACTTCCAGAGAT
ATCCTACAAGCTCCAACTGCTGGGAAAAGCAGACTGGGGTCTTCCTCAGG
CTCCACCAAAGATAGCCAAGTGGCAGCAATGACATGAGCACCTCATTCCT
GTCCTAGACCCTGGCAATTCCATGAGAGGGAGGCTCTGAATTCACCTCTG
TTCCCAGGATGTGTGACATCAATGCACATTGTCATACCAGATACACCTGC
CAAACTTCAGAACCCCTGGGAAGTGACCAGAATGCCACTGAATTCAACAC
CTTAGACCAATGAACATGACCTGGGTCCTGTAGGATAGGAGGCAGCAGCA
GTGTATACCCAGACCTGGTCGACTCGAATAGATCTGCTAGGCGCCAAGCT
TGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGA
CACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGA
GCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAA
AAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAA
AAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTAA
CTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCC
CTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAA
ACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTA
TTGATGATGTTT
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.
SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> IMPROVED HELPER DEPENDENT VECTOR SYSTEM
FOR GENE THERAPY
<130> 20377 PCT
<150> 60/118,601 <151> 1999-02-04 <150> 60/138,134 <151> 1999-06-08 <160> 17 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> Consensus sequence <221> misc_feature <222> (1) . . (15) <223> n = A,T,C or G
<400> 1 atttgnnnnn nnngc 15 <210> 2 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Adenovirus 5 <400> 2 attttgtgtt l0 <210> 3 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Consensus sequence <400> 3 attttgttgt 10 <210> 4 <211> 158 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic packaging signal <400> 4 gtacacagga agtgactttt aacgcgcggt ttgttacgga tgttgtagta aatttgtcta 60 gggccgagta agatttgacc gtttacgcgg ggactttgaa taagagcgag tgaaatctga 120 ataattttgt tgtactcata gcgcgtaatc tctagacg 158 <210> 5 <211> 158 <212> DNA
<213> Artificial Sequence <220>
<223> Adenovirus 5 <400> 5 gtacacagga agtgacaatt ttcgcgcggt tttaggcgga tgttgtagta aatttgggcg 60 taaccgagta agatttggcc attttcgcgg gaaaactgaa taagaggaag tgaaatctga 120 ataattttgt gttactcata gcgcgtaatc tctagacg 158 <210> 6 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> Linker <400> 6 agctcggccg attattggcg cgccagatct gcggccgctt ctagaaacgc gtgaattcgg 60 cgcca 65 <210> 7 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> Linker <400> 7 agcttggcgc cgaattcacg cgtttctaga agcggccgca gatctggcgc gccaataatc 60 ggccg <210> 8 <211> 40 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 8 attggcgcgc cttctttctg ggatgattcagcatcaactc 40 <210> 9 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 9 gatcgtcggc cgcttgggtc atagacttctttgagaacca g 41 <210> 10 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 10 atcagttagc ggccgcacaa gctaagatcacaaagctgtt t 41 <210> 11 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 11 tatggcgcgc cgctgacacc cagcctgggtgccggtg 37 <210> 12 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 12 tcgacgcgta gcgctgtgtg gccttggcagtttccatag 39 <210> 13 <211> 45 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 13 tcagtaatgc ggccgcggga tcattcctgg actcagattg ttctg 45 <210> 14 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 14 tattaaggcg ccgggcatgg gagtgatctc accaactctg g 41 <210> 15 <211> 46 <212> DNA
<213> Artificial Sequence <220>
<223> PCR Primer <400> 15 tcgacgcgta tttaaatgtg ctggagtgtt gagatactgt agtggt 46 <210> 16 <211> 28068 <212> DNA
<213> Artificial Sequence <220>
<223> Modified adenovirus <400>
aaacatcatcaataatataccttattttggattgaagccaatatgataatgagggggtgg 60 agtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaag 120 tgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgttt 180 ttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttg 240 tagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataaga 300 ggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgc 360 ggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcg 420 ttccgggtcaaagttggcgttttgattcggccgcttgggtcatagacttctttgagaacc 480 agttataagctatggtttctctccacagaaaaagcacttatggtgtctccccctttccag 540 cccaccaacattttacatctaatttgggggggttttctggaccacttaatacccatccat 600 ggatctcatgtgaagactcccctggcttgagaaatcactgtcttgttgaaaatgggaaca 660 aagctaagtcagatagctggttcatcagcaatgactttgaccaagcctgatcccacccta 720 ccccaccccaccccagtgaccaccccccacaatggagcacacaactctaaactggtttgt 780 aggtatgtgtgtgtgaacacgctgaggaatctgcaaaaccaaatggtgagtgcaaaacca 840 aacagtcacgagtaaatctcacaacaaccacgtcctgagctgcagcccttgttgaactat 900 accccactagggccccaagattttaggacttgtgtgtgggtgggacctcccctttctatc 960 atgctttagaagacagggatttcaccagaattgaacatattgaacatatgacccattttt 1020 ttcagccaaaggcaattaaaataacttcatacttgatatccatgtcagcaaaagctgcaa 1080 aacgcaaatgggtggctgctaagagccctggtaccctgacgagcacaccaagtgcttagc 1140 aacagtggtgtccaaaggaccagctggaagcctgccttgatgagaagttgctcttctttc 1200 tacatgaaggaacacctctactctcctgcttttaatacctgagctgtgagtgatcatcta 1260 tgtccattagcaaacatcccagtggagaaggaaacactcatacccgaaatctaagctaca 1320 tagttggaatcacttcaacttattgcaataaacacttactaagcacctattgtgggcaag 1380 tctttgcaatggataatagttcagtagatattttgatgtaatatttgaaataacaataaa 1440 aattgccaccactgaatttattgagcatttgctgtgctttaggcactaacccaggttctt 1500 taaatatttggtcttattcgatctgtataaatagccatctatgagaaagggactattatt 1560 gcccttattttacaaatgaggccaatgaggcccagagaggttaactaagttgcccaaaat 1620 catacagcccactagtggcagagcaggatgcaaacccaggcttgcctcgttcccaagccc 1680 acatgtcgtttgcattggtgtggaggtgtgcatgtgtttagtcattagcatgttatatga 1740 taagcaagttttgaaacatagaaacttaaaatgtgccattaagaaaagtacaggcaaggt 1800 tttccaaggggaggtgtggacctccggacaaatttttaagaactaattataaatacttaa 1860 aaatgggaataagaagacaacctaactacctgaacagttttagagatgactcatgcccac 1920 cctctaaaacccaaacaaaaacaacaaagtcaagaaaacccatgaaatcttagcaagcga 1980 tttctatgtacttgtgaaaaggatttctttaccattctaatgggatttatgccaaccata 2040 gagggctcagtgcccctcccatggggtggttagtgagtacagagctgagctcaccggcca 2100 tctgcagcttcatgttatcaagctccagtttgtccttggagcaaggttatctgggacatg 2160 agcagaggcattgctttctgcaatggacagttctttctgcctgcatacctagctccttga 2220 taactttaaataccattttatagccacactggagttttgaagacctcaatatgcaaatat 2280 tactcaggttctgattacttgtctgctccatgataacacactctaaaagcaatgaatggg 2340 gcttatttgtagagaactgaagcattttaagcttttgctcaggaatccctggtagcttcc 2400 tgtgacttgcaagatattagtgatgggtcaagaaacaggaccccccatcagcataacata 2460 cgcagtgcctcagtagtccatcaggcagaaaaaactgcagatggcacatggaaatgacca 2520 gcggcggaagataccccgacagtgtgggcagttctatttcagcagcaatcaagagggggc 2580 ctggagccactcaatcaagtggagcaggatgggagcaagcactgtgcagaccaatgcaat 2640 ccccagttaacacaaaaaataaataaaagagatgagattcagtctcttgactgtgactga 2700 ctgggagctttatagctgatgcttgtgtcttttctccattttatttaattaggaaaagaa 2760 atgcttatcacacactctacgtgtgaggtacaacctccacaggaaaggttgttaggaaca 2820 tttcaacttctagaagtttctaaacataaggtaaatccatctttgtccttgggatcactg 2880 cacatctcagaaaggcaaataaatcagtaattggtgggcataattactagctcatggact 2940 gacaaggtctacactatttcgaatctcacagaagtaagccatgggacagatagagtctga 3000 tagtggtgccccgtttcctggaggtcacacttactcatccccctggaccctgggcttctc 3060 atgattgtcagagagtttgctggaaccaggtcagcccagtttcccttcccctgaaaaatc 3120 ctccaatggctcgcaccaagactagagatgcaagtgcaagcacatccaccctctcagcag 3180 ccaggttttgcgttccataatgtcacgtacccccagtcataccaatctccttggagctct 3240 ccagacaggctgccatgtgtggtcggccctctgtgcttgtgctccttggtttgccaagcc 3300 tggaatgccctttctccatgattgtactctgggaattctgtgtgtctttcgagatgaagc 3360 tcctccaccctggaaattctgcctctctttccaggtccagctcctgtggctgaataccct 3420 tggctgactgaacattcttgctgggccaagtcttaatcatctctaaatccctctggtgcc 3480 ccgacagtatctagcccaggcaaagggctcagcaaatacttgcaaaattcaataaacttt 3540 actatgttactagcttggcatgatgttctaggcacttgggagattataatctggtgagca 3600 ttgtgctagtcttttttttgggataaacaccaatgataaactgagcctttactctcattt 3660 tagcacatctactcatctcttttcaatgctggtgggtttataaaacccatcagtagaaca 3720 gagagtgatcataatcatatggtgaaaataacatcagctaacatatttactgaacatgct 3780 ttagtgtgctgggcactgaactctgtgcacatgtgaatttgagatagattgttcctagct 3840 aataagatgaggaagtggagatgggcttactcaagtcacacaatagcaagatggggtaca 3900 gacagaaaccaagctagaaaccaagcaacccctgggtttggaaatgcatgggctcctcct 3960 ctgcacatggcgaggagcagtcaggtgctcctccttctctcatactgaaataactctgca 4020 cttttggctattctgtgacactctgtgctattctgtagctcaaatggctctggtgcaagg 4080 agctcagattatgactaacccccatagatattcagctgctttgcaagaagtggatgaatg 4140 ctctggctttataaattattgactagattggatattggcccaatctccactctggtgatt 4200 cggaaggaggcatatgcacatttgcaaggttatagtagtgcactctaattccactggctt 4260 ctgagagcttgtaggtttcttgacttaatcattctggaattaaggtaatggatcctcaac 4320 accttttctttccctggcctttactaaccatgtaacagaaatgagcagagaaaacccaag4380 aaagcgaactggagacttgatgagtgtgtcaaagatgctcagagtccaaggtcctctgtg4440 gctcactgacttcagaagccaacctccgttgttcaagtcacttgtgaggttactatgcta4500 gagcactaaatattatccagatagcccaaagaggtgaaggcagacatgtggaagaacctg4560 gatttttgggcaagttgatgaatgcctcctgccccatatcaaagagaggtgataggagac4620 aactttgtgaatttgaaataatgcaccggggaaacaggaaaacgtaatgtaagtcacgct4680 tcttggcttttttcttgccattaccctacttggccaagtgcaaatgggatttcaatatat4740 cataagtatgcatctattaataacaatgcaagaaagctgtacaactgaagtctgagattt4800 tgtaagaaacagaatctctgtaagcatcaccatccaacagaacttcctgagttgatgctg4860 aatcatcccagaaagaaggcgcgccagctctgcaggatcttcaagtctggggtgccacca4920 gcaagcgacggtcctccatgggctcttcaccttacggcagtgtccagaggcaccgccagt4980 cctctgctcctatgctggtcctgctgtccctggcaaaaggagccagagcattctctccag5040 gcctcccgaggaggctgcttcctttgttttgcagatggaggctcccatcctttgttctga5100 atcaatgtgctccaaagataagccccaagaaaacagttgttgccttttgacactgacaat5160 tagaatcgttggaaaatggagaaaacaggaaatggcaaatggtttcagtgaccaggagga5220 aaccgtgcctgaaagttgctgcttagtgactgggacactcgctttctgctctcttatgaa5280 ggacagcctaggccgtgtggccttttataaacaaagctatgaaggggtcgtcaaattttc5340 tagggctgcaactgtggcactacgtcctgttgtgccaggtgacactgacaagcagcactg5400 agttctatgcaagcccaggtgtgcttctctcatggtgacccccagagaactaaggcccag5460 ctcttcctctgtcacacccctcccagcccccactgtcagacaagggaccacattcacaga5520 cagtctcagccaagatggcaaccttggaagtcctggggatgcctttctagaagctttagg5580 ctgacgccagggagctgtaaagcccccacctgtccctgggggttgtgtgctgggcaggag5640 agaggggaaagagcccaaactccaccactgcctgctggcacaactgagcccacgccaggc5700 actgcaccagcttcactcaccagctaccacatgctaatgtctcctgtttacccaggtgca5760 atcacctgagtgctgctttctcaccactgcatgggaagaacgatctcattaccaattcaa5820 ccacttaagcaggcaaaaggactccaacgggggaaaggcagaggacaaacgattcgggga5880 ctgagacagatgcccacaaaatgttcctggattttagccggattcgggctgaagttcctt5940 gccctatgagcccttggaggagagcatcctgactcagacaagaattcaaatgacaagact6000 tctgggggatggcagtgaatggatccaaagccttgttttataaagaatgtggaggaaggg6060 aggaaggaagggagggaaggggagactgagagggagagagaataaagaagaggaagagaa6120 ggaagaggaggaggaggaaggggaggggagaggaaaaaggggagggtagaggaggaggga6180 gaaggggaagggaggacagaagaggggagggagcggggagattatctacccctccatgct6240 agggaactcctgtctccttttccagcagacactgctccatgagtgcccccacagagagcc6300 agcctgccacacagcagaaggtgctcagctatctgtcccaaaggttgagcactagtgtat6360 accacaaggaaaaaaaccttcttttgtactgtttggaagccagaattagaaacaactcgt6420 tcacccttctgactttccctttgcaatgacaggaagatagtatactgcatgggaccacca6480 cacattcaagatctgagttgtggggacccccacccccacatgggtggagccccctgagac6540 ccgtgggctctgactggccatggcccatagatagtgacaacatacacactgctatctgaa6600 gcctagttggaggacggatggatgcttttaggaaaaaattccctttagtcgtccctcaga6660 aactacaacggcttcctaaaatgaatggcaggcaagttttgattgtttcctaagttcccc6720 ctaactctcagagcctgtgactcagcttctccagaatgcctgcagggaggcaggaccact6780 gggagtaatgagcacgaattgggccacttctcccagtggactgttaacgccgagatctgg6840 ggtggaacccactgaacacctgcccttttgcgttgcccagaatgcagccagagggatacg6900 ggccacagtaactctggggcccctgaacctaccttcccaagaccctgacgggaaggtatt6960 gcttacatgacagtccagtcagtccctctgtcactcagttaacaaacatggtcatgggtc7020 tactatgggccaggctctgttccgggcatggggataacaccgtgtgtctctactcttatg7080 gacacaagtaaatcaatcaaagcagataatgttctaatgaccacccagtagggtgaaggg7140 atggagggtgggcaggtgctggcagaaacctaaggtcaagtcatgggaccccaatgggga7200 actggaaactgccacaccattttctatgttaggggtctttccttgcccttctagagacct7260 cacccatggtaatgatgctgccctcaggtttgaactggtctgaagctgtcactgggggct7320 tcacaaaatgaatgccactgaaagcaaacggcagagatctgtatatgaaattctttttac7380 cctttgttttctgtcttcccacatgatctatgactgctgcaaactctattctgactcctc7440 ctcagtcttggccggtctgggaagaacttggtttagatgctgggaatgatcccgggcaaa7500 ggaaacaaatatatttccagcttgcttttcttcttcccctgtggtgcctgcatcctctgt7560 ctcgcactgactcacaggctggactgacaaagtggctgtgttgacagggaggatttagtg7620 gatgccaggcctgaccctcaaaggccctcaggtggggccaatttccaaggttcttagaga7680 agatttggggccacaaatctgctctgccacttcactgctgggtgaaagctgttggcttga7740 gaagagagagaataggtgagggacaataattctcaaggcaagctagctgatatcctttca7800 tttgtggcttccaaagacagggaaaggcaaggcagaccagggtggaaacagcgacatcct7860 gaggggacgacgtccgttttgctaattctgtttccctggcagctttgtgctggacaagat7920 ttctctggtttcagctgaagagtgaacagctgaggagctctgtgactctgagagagagag7980 aaatcaatagcccttgaaaggaaagcactgcttgttcttccaggccagggcaggcaggat8040 aaagagaacaaggctgtacataccaaagccgaatttccattaaaggccagaatcctggga8100 ggccctttcagcttaatgaccatcttctgatcactatcacttagaaaatgttcatgtctc8160 cagttttctctgcagatagtgcagacatcctgtgcacattgatattttatgcaccagatg8220 caattctctgcaaaggtcccctctggctggtgtccatccgcttgctctcatgaccactga8280 tgcgactctgggggcaagtcaagccattccttctagactctagaatctagaggatgatcc8340 caactcacaggcctcttggacaagcttgctttggattccttctgcctgtactacccagac8400 tgccagtcctccagcagggcaatccaacttcctgcctctagggtgtgggagtcctgggcg8460 gggtttggaatccacagcagatggctggcaggagaggcagttcccgagcacccactaggt8520 gccaagcaggcaggtatcaggtgcagggcttcggaggtgcagaagcacagcccctgtccc8580 cctcaggttcccagtctggcctcagggacagacaggcaggtaggtatggaaacggggtac8640 tggaaggcatacagctgagggagagagcagctgcaccctggtcaatcccccactcatttc8700 cctggaggtggggctgaggcacctggagcactctgcttgcttgggttgtccctttgccaa8760 agaatgccgggcagaaagtagagaacaagcaggcccaggaagcactagaagtccctcatg8820 ggtggacagagctttaccttcggagctgtcacctcctgtgttcctcccaacaaccctgtg8880 aggcaggcgggcaggctgagcaggggactgtgccccgttgacagatgaggaaagcgaggc8940 tcagggggaagtgacttacctgtggcgtcgcagctaggcagtagtggagcgggcacctgc9000 ctccaggccttcggatcccagctgccccagggcgctgcctctgaggtgctgtacgaggag9060 gtgactcgggccagctcatgagcaaaggagctgccgggcaggaaggactcactccccagg9120 gcctggcaggacgggggctgtcgcccgaggctgacctgggggtgcctggcttccccaagc9180 ttgcaggctttgtctacatgagtctacaacaaccgattgaacaatccatcagctgcttgg9240 ccacaaaatggttctgactgatcttgtcactgccctgagtgtctagctgggtcatctggg9300 tgttgtggttatacccaccaactgctttggtggaacgaaaccgcggccgcgggatcattc9360 ctggactcagattgttctgaagaagcccagttctgggtggcatcaagtgcttgctagatg9420 gggggcttgccttgatccggctacacttggaggtgacttgttcttggacggctacataca9480 gaaagagagaagtggggatgagttccaaaggcatcctcgacttcggctgtggccaccgga9540 gggtagctcctggcccaacacggacttctcacctcccgcccttggctctctactgagctc9600 ccccctgctccccaattcctcgccattcccctcatttctctgccctcagcctggactgca9660 gttcttctgggaagctgccccaactccctaggtctgtgctcaccaagagcagatcacact9720 ggactgaaatgccagctgatttgtctcttcaagaaaattggaagctcctggaggtcaggg9780 tccatgtctgcttttacactcagtgctctgtatgcaggcctggcactgcccaccctttga9840 caggtggtgcatattttgtagaaggaaggaaggggccaggtggggtgggctgggctggtg9900 gcgggagctagctcagcctcttagattctctacccgatggatgtgacctgggacagcaag9960 tgagtgtggtgagtgagtgcagacggtgctttgttcccctcttgtctcatagcctagatg10020 gcctctgagcccagatctggggctcagacaacatttgttcaactgaacggtaatgggttt10080 cctttctgaaggctgaaatctgggagctgacattctggactccctgagttctgaagagcc10140 tggggatggagagacacggagcagaagatggaaggtagagtcccaggtgcctaagatggg10200 gaatacatctcccctcattgtcatgagagtccactctagctgatatctactgtggccaat10260 atctaccggtacttttttggggtggacactgagtcatgcagcagtcttatggtttaccca10320 _ aggtcaggtaggggagacagtgcagtcagagcacaagcccagtgtgtctgacccacccaa10380 gaatccatgctcgtatctacaaaaatgattttttctcttgtaatggtgcctaggttcttt10440 tattatcatggcatgtgtatgtttttcaactaggttacaatctggccttataaggttaac10500 ctcctggaggccaccagccttcctgaaacttgtctgtgctgtccctgcaactggagtgtg10560 cctgatgtggcactccagcctggacaagtgggacacagactccgctgttatcaggcccaa10620 agatgtcttccataagaccagaagagcaatggtgtagaggtgtcatgggctacaataaag10680 atgctgacctcctgtctgagggcaagcagcctcttctggccctcagacaaatgctgagtg10740 ttcccaagactaccctcggcctggtccaatctcatcccactggtgcgtaagggttgctga10800 actcatgacttcttggctagcctgcaacctccacggagtgggaactacatcaggcatttt10860 gctaactgctgtatcctaggccaataaatgttgatcacatttatagctgccatggtaggg10920 _7_ tggggacccctgctatctatctgtggaggctctgggagcccctgacacaaactttctgaa10980 gcagagcctccccaaccccttttccattccctatacctgacagatggcccaggaacccat11040 tagaaatggaaggtcactgcagcagtatgtgaatgtgcgtgtgggagaagggcaggatca11100 gagccctgggggtgtggcagcccccaagtgattctaatccagatcctagggttgtttccc11160 tgtcccattgaaatagctgctttaaggggcctgactcagggaaatcagtctcttgaatta11220 agtggtgattttggagtcatttagaccaggccttcaattgggatcctgctcttagagttg11280 gatgaattatttaactgattttcagatctcctctttctcaatgctttcagaagcacagta11340 actgcttactctgaaatgaattctcaccccacttccacatatgcaccccttgcccacccc11400 tttgggaacactggccttaactgcttaccttcaaatggactcatctgttgggagatatat11460 gcattctgccgttcaggggtcattgccataagacctgatctctgttcctcttgctaaaca11520 gaagatgaaaaagacaaattagattacagctaccaattaataattagccttaggatcgct11580 gcgtggggacctaggacttggctttggtgcagcagaaagcatgaataaacacaccagcat11640 acactcgcatgcatgccccaccctctcgagcaaaattccacaggtataaataaagtaaga11700 ttctgcacctgggttaaaaacacaactgcaacagcatagaatggggcaggagagacagaa11760 cttaatagcaagagcacacagaaaaaagttttaggcattttggatgtccatctgctcagg11820 atgggtcagcagtgagatgcggtcaccaaaagaacaaatgtaacattaggctgcattaat11880 agaagcagagtatgtagaaggagggaggtgacagtcctatgctaactctgccttggccag11940 actatacccacaggagtctgggcatgccagtctcagggagacccagacagactggctgca12000 ttcagaggatggtaagtaatgagagtggggattggacttcaaactacccagacaaagaat12060 ggctgagcaagccaaggatgctgtggctggggcagagcagactgtgggctatgtagtggt12120 ggatacctagcctctgcagggctgtcatagggaaaggacattgagaagaggactgaggct12180 tgttcctggtggtcctggcatgaacggccagatgatcacatggtcaggtggacacagtct12240 ccaacactgggagtagccaaacacttactgccaacctcccgcccttctcctgactagttg12300 cagcataggcaattgggaggagcttcctgtctccatctgaaagctggctgggtgggcagg12360 gggaggagcgagccaagtttcaaggccgcagtttcagcactcagtctgggatcggctcaa12420 ggagcaaaggggaagaacatagccaggagggaataacatgaaggcccccagacccagaaa12480 aggcatgacttgctctgagaccctcagccggttggtgtcaggttgtgactcggatccagg12540 tctgactcccagtccagtgcttgaagcctcaccccacacagtgaggggagcccggccatc12600 tctgctcaactgctgccatctctctccccttctcaaccaccaaggcagctctgtctggga12660 gcacaagctccaagtccactttctggtctgtgtcccccccaagatgccagaggacttgcc12720 tctacaacacgggctgcccgtgcagtgcctgcttttccagcaaagggcttctgggaaccc12780 ttctctgcactcagtggggctggtgggagtggggcggggtagcgacccagtgcttgggac12840 tgtgcccagctctcaggcctggcagcagttcctggccttggttcctgccaaggcagagag12900 gacaaacacatggcaccgggaagactacaccagaagcgattccaccagactggggtttgc12960 ttttctatcccgcccttagcctgcttcctgtcctggtccctgcctccccctccactggag13020 ctgccgtgtgggcagtgaggggctgtttctcagctgccctatggagctgccctctccctg13080 ccaaagcattggcaaggcggcaaggggtgggggtggggatggggggtgggatctgccttc13140 tcaagctctcattatactgagcacgtctcacccattattttatgtcatctagcaacaccc13200 catgtggacactgaggagcatgggggtcacatgaccactgcccaaggccacaccatccgg13260 atctgcctgagatggtcagggttggcagccatttctgaaggcagtcctttcgctttggct13320 cttcttgtaccagtctcaggacatcagggcagaagatctacagtccccagcttactgatg13380 tgacagcagaggctcagagaggttaaatgacttgcccaaggtgacacggctaagaagtac13440 agtatctcctaactgcagaccaggtgcttctgctgcttctggggacagattcctgcgtgg13500 ctggctaggtctaaacggtccttaactccatccccaccggttgctgcattagtttcatca13560 aataacacagttgtacagaggtaggggttcaggggcaggggcagatggaggctggagagt13620 gtgactaaggaaacagcaggggaagtgcggtaaagtccgaagggagggacggaaagagaa13680 agccaagcccaggggcgtgccagacaaaaggaaaggccacgccggggcagggcaggcttc13740 agcgggtgctggggcgtcttcatcccgggaagcacacattccagaggaccccggagtcta13800 atggaaaagctggccagcctatcactatggaaactgccaaggccacacagcgctacgcgt13860 atttaaatgtgctggagtgttgagatactgtagtggtgggatgcttaagtgctggggtgc13920 tgggtgttgggatgcctaggtgctggggtgccgagatgctggagtactagtgtgctggga13980 tgctgaagtgctggggtcctgagatgctgcagtgctagggtgctgagattctgggctgct14040 ggagtgttggggtgctgggatgctggagtgctgagatgcttggacaatggggtgctggaa14100 tactatggtgctggggtgctggggtattgagatgctagggtactgggatgctgaagtgct14160 gagatcctggagtgctgggctgctgggccacaggctcttgaatccattcgtctgcccagg14220 _g_ ggaagaaaccagaagataaagagctaatgaaggagctttggttgagagggaggaagtaat14280 ggaaggagcaacatcttgtggaggagcaggagagaatggacctcaggttgggagagaggg14340 ccaggctacaggccagagaggcagaaggattccagcagagtgtgggctccaggagccaag14400 gggaaacaggtttctgggaggagagagtccagtactgctgaagtggcaagtccgctgagg14460 accaggaagcttcatttggctttatgaccaggaggaatttggaactgtgactagagtact14520 tagggggaaggaggcaagactggagccagattgctctgggttgaggggtgagtgggaggt14580 gaagcagggcactgtcactcctttgaagggtggcagagagctggaattggtgctggatgg14640 gctgtggggtgacagggtcatgtggaaagcccctggggggcacctggaaaaggagaagct14700 gacagtacagtgagaggacagctaagggaaagcggaatggcagaacacgcactgccagga14760 ggaatgaggatagggtcaggagtgccaggggcagtgaggccagcctggggtcaggtggca14820 ggacgtgtccaggaagctggtctgcactgcagcccacactggctcagccttgaggttccc14880 tgtgtggttggggtaggaagttgaaccctctgggaatggaagatggaaccagctctgcga14940 gccaagctcagcttttatctatgggtctctgagggctggcagagctgagtggggacaact15000 gtgatccgtgaggctctcaggttgaggtggcccctccgggagggcttcattttcccagcg15060 ggtaggttctaagcagcagtggctgggcaggtgggtccaacacagagccagagaagggtg15120 aatgggcctcctggcaccccacccctgctgcccctgagctcagtgatggagggggacagc15180 acagctgagcccaagtgctttggtgtggccctgagggaaagctgcagcctgcctggggcc15240 tggcatggatgggacacttgaggcagagggacaatagtgggcgctgcagtgaggctggct15300 cttggagaggtttcctgaggagtgctgcctgagacgggcagggagaacagagacaaagtt15360 ggtgacagggaatgaaagctgactgaaggactttacccagacctatgaggatatctctct15420 cagcaggaagcaggaggggactgtgtgaggactggccaagagctggagtgttgggaaaat15480 gactctttctccgacccctctgtcctagctctggcccctggactgcggaggtctgcttcc15540 acccccattggtcgatcgttgtcccttgtcacagccattgagaattttggcagggagcat15600 gttcttagagcatttttaggctctgcgggacataacagctctgcctcagagcacatgcct15660 ttctcagctcctgaaagccactgatcaaattggaacattttgtaccttagggatgaggat15720 atcaactctcccagccacttagagggataaatgtgatgatgcattcaattgtgactacat15780 ctgatcccaactgttgcttcagctgctctcctatagcacatggcgggaggcgtgcatccc15840 agtagctacctccccacttttggggagatgtggttccatccatgaaacctgggtacccgc15900 ctaccaggtcctggcctatcaggtggcagggtctggtcaaagaagggcatgtgtggtctt15960 cagcaagggagacaggacggtggtgcagagcgtctagaccctcagggcaagtctccccca16020 cacctgctcccggggcagttgtctttgtgacctcccatccccctctgtttcatcctctat16080 aaaatgaggggctgagccccaaaataacaggcttctttgccatgatgcaaaactgctgaa16140 tctttctttctgacacacaaggcatcgagcagcctctgaaagaaccaaagccactagcag16200 gcttcctgacttgggtttgtaggtactgaatactcccttgaaaaataaaaacatagaggc16260 acttttctcctggctgtttattacagaacgaagaaaaaacacactggcttgaaacagacg16320 ccagatttcaaatgtagaggtgaaatacgaggtggcaattaaaatgtgattacagaaagt16380 ctggacactgagaaaagtttacaggacagtgggtgtgggttttctataacagacacttaa16440 atatacatgacgataattgcagatagaaaccatcaaagacaaaccccaaatcaactaata16500 atgtttacagatgttcccccccaaaccacagagccttacatcaaaacaaatactgaaagg16560 ctttaaaccaggaacagctcgccttaaccccacgagggtgcacacaagctgggctttttc16620 tctcggtctgaatggtaaagggaggaggatactctagctcctccaggtggattgctgaga16680 cagggctcggctcacacactgtctctgcgcctctcccaaatctggagaactctcccagcc16740 tcctggtaaagtgtctctgtggggcacttaacgataaaacagcttctgctgtaaagctca16800 ttaggaaagagctagcggagactgaaaggttcgcaaaagagattaagaatcacacaaggc16860 aataggatttttagtgaacatagaaataaatggccaagtggttttctatttggcatttgt16920 caacttgcacaacaactcttggtcatatccacattgctcattgcattaaaaccataagcg16980 actcagccacctagcttaacaaggtatcactggagcaaacaacacggtctgcatatttgt17040 aacattgtataataaacacaaaacaatgcatagtaaacacaactctactgaaacaaaagc17100 cgtcgctttatttacaaagtcacaaaatgaagtataaatacttctgtcattaatgtttag17160 gaaaaccatttacaaaattttcaaatatgtacacgtagcttgaaaaatcaccagctttcc17220 attttgtcacaggtagagagagggataagcatgggctgacaacaccactcaaattgtaac17280 gggagacaactgcgggtatggatcgacaccacttcctagagtgatgtcaccatgggggtt17340 tctatgggcatcctgctcagatttaaagtgccccagcatcctgggtgacttgcccagaat17400 tctgggctgtggcattttgagcagcagcatgctgttccaaaatgtcgtcgatcagcctca17460 agttgcacacccagtcttcatctgggctcacacaggagcctttcaagagagcttcaatga17520 aatctacctcattgcagtcaggtgacgaaatcagatcatttagtgggggttggggctggc17580 gcaaaaagtcggcaggtggcagctcagggggaatatccgttctgtcgaacggacctggga17640 actggctggcagcaacggcagaagcagcagcagcggtggcagcagcagccacatagcttg17700 gtggctcgatgccctgtatggggctcaggggactaaagctggccataccctgctggagga17760 acttggtggtgtttgctacaggcaccgggccctgtaccgggctctgcctgaggctctggc17820 tgcccagcaggctgaagctggggttgttggccaggggcacttgtgttcccatcgcagcgg17880 gcacttgtgcctcccaatcagatggcctctgaaggcaggcctggccagaaggtgagtgct17940 gctgaacgctattatccacttggctgaggggtgttttccccgaaactgctgtggtcacag18000 ctgctgccgctgtgacccatgcagcattgttgaacgcagtgggcattcttggcacactag18060 gccgtctgagctggtggggactcaaggactgggtgcccagggagctgggacagaacccag18120 gcaggggcacttctggtggggtggccttggggctctgcatatgctggcagacagagtcaa18180 gtctgcccaggggagtctggcctgagtgtgagaggatgggacactgggggctggaggtga18240 aaattccttgccgcttccccagagttggtgagatcactcccatgcccggcgcgcgcgtcg18300 acacgacagggacatactgggaatgcgcccttgccgtggaggcggggacccggcagcgct18360 acgtatccagcatcaacctgtatccagcatcaacccgccaagttcactaacttggtaggg18420 gtgaggttagggatccttaggagcccaggcagccagactttctggggagcccattcccat18480 ttgtgttgccaaagtacccccagcaggttgtgggaatgttgcctgtgaagagagtctgtt18540 ggggtgagatcttgtgtgtgtgcacagggtgacagttgtgtcccatttcccgggaagctg18600 tgatggcagcagaacctagaggagcctgagagagtgtgggagagtgggcctctggaagag18660 tagaggctgcggagccaggtgcagggctgtctgtcacccaaaggaagagggactgatgac18720 tcactgagcgtgtgtgtcccctggtggcagcaggccccatagtgaacataccataccttt18780 tctgtcctgagcgatgctcccagcagtcctgggagatggaacggtccttattcggctcac18840 aggaaggaccgccttaactggacagacacagcaaggtgctaaagatgccttccatcagag18900 gccaggttggaagctctaaagagacttctcttgctgttctctcacccacccccaggttgt18960 gtgtgtcccgctgtggattctcatgtcctttctgtgcctggtggtcctctactacattgt19020 gtggtccgtcttgttcttgcgctctatggatgtgattgcggacagcgcaggacacacata19080 accatggccctgagctggatgaccatcgtcgtgccccttcttacatttgaggtaagcgtt19140 ccacgggaagcctcttcagcccctgaagcttgcgcttcccctgacaggattctgcacccc19200 tagaaaggcagcctctgtccctcgagctcacagtgagcccactccaggagaggggagaga19260 acacagccatctccgagagggagcttcggtgaaaggagagcatccttcctttctcttggg19320 ggcagcacgtggggctggcagggagaagagtgcacctttttagccatggtgcctctgtat19380 ggctccagtttccactctggggaaagcagagtgggatgtcagatttgtgtattggagtca19440 cgtggagaattctagaatgggagctgttgactccttagaacaaacacccggaggagtttg19500 ccataaaactgctggcactgggaacttttcaagtggataggctattgccgagctctgaag19560 agggacataaaagctcatttcgagctttccccagggataggtggtttcctgcctttttct19620 ggcggtgctgatgttccctcttgtgggagctcacgcgggggtggggtggtggggaggaac19680 tgcctaatgaagtctggcttccgcctctgcccattttcggtgctggcatcaaccgggact19740 atgtctctttctttagattctgctggttcacaaactggatggccacaacgccttctcctg19800 catcccgatctttgtccccctttggctctcgttgatcacgctgatggcaaccacatttgg19860 acagaagggaggaaaccactgtatgtactcagcatttcagaagtccttggtgtgtgtctg19920 gggggggaccagggggtggggggtggcggatagaagtctaggaagggatgagtccccgag19980 ggccccaatttagaagcttgtgtgggaaagtgagggctgaggaaattctgggaccttcta20040 agggaagggcatgccgtaactctggtgttctgctggcctgcaccgggacttttctcgcag20100 tgcacgctgccatttgaggtagaaccagacacggcaggcaacctctcagagatcccgttc20160 cctcctctgcaaaatggggatcaagacagattcttcccaggcccgggagggtttgatgga20220 aaatccacatctcccacccaaacctgggattcatcctaggtccctgttggccgctctgcc20280 tcccccatatccttgctgccatcacccgagtcttgcctgtcttgccttgctaacactcta20340 ttcccctccacctgcttgctgaggcagacacttccaaaacgatctctgcagagggtgcct20400 tcctggcaaggctgtgggctccatggcacggaagcccagagcattgcccttcggaaagcc20460 agtgggtttgggggcagggcctcactgcagcccagcagcccgggctgtgcttgctgtttg20520 tgcctctgccccctaccccgcacccgggagcagggagggcttgcaccgagctgacactcc20580 agtagcctacagagaggagtagtgggactgggaaagtggctttaaggtggctccatgagt20640 tcaggccccctcctggccaacccgtgcatgactaccgccctcacggattccagagggtga20700 cagaaatcttgttcttgggtggcactgtcatccatgagtttatcctggctggagaagatt20760 agcggaagacaccgtagtctgcgcaccacagatattttgagactcactggagcagtagtt20820 ctcaaatttgggcatccagcagaatcccaaaagggccaggaaaaggggaccgctggagcc20880 caccctagcccgactcagtttctggaggtctgggctggggcccgagaatggcatccctaa20940 ctaggccccgtggacgctgtccctgccggtccgggaaccccactccaagcaccacagagc21000 tagcatttgcacttcttccccattttgggtactcaagccctgttcaggctttgtgactca21060 ggagtctggataaagtatgttatgacattgtaggagtgaaacttcttgttacggaaagaa21120 agttaacaggaaggtcagttgagcctcgtgtgtgaaataaaaaattcttatttttcaggg21180 tggtttggtatccgcaaagatttctgtcagtttctgcttgaaatcttcccatttctacga21240 gaatatggaaacatttcctatgatctccatcacgaagataatgaa~aaaccgaagagacc21300 ccagttccggagccccctaaaatcgcacccatgtttcgaaagaaggccagggtggtcatt21360 acccagagccctgggaagtatgtgctcccacctcccaaattaaatatcgaaatgccagat21420 tagatgccacttccggggacagagcttaagtggactgggacgcactctctccgccttcct21480 ctgccccctcgttcaccccgcagaccagaaccagtactggagctgggtctccaggtacgt21540 ccatctcatgccttgtttgcatccagcgcctatcagccactcaccacgacgggacgcgga21600 agtggcaggtgacgggggtgtgtgccagcagatgcggatgccaggaagagtgtgagaaca21660 ggggtgggattaccgtctgtctgggaggggctccaggtacccctcttccccgtcagaccc21720 actgggagatggctgcttgccaggcccccagaaggaacatctgtctatacggtgctgaaa21780 tcccaatcaaaagtattgtttagaaatgtatttctccacagggctgacctcctgcagctc21840 gctgagcactcccaggtcctcagcactcccaggtcgtggctggggcagtcagtaggaact21900 gtaactatgtctctgatgcaccacgtgtttagacacagcacagtccttttttctgttcct21960 actgtggaagtagtttctctttgggcatgctgacagcagtttttcatagcctcacggatg22020 agccctttctacgggagtgactccatgcttgtatacagagtatttatacaaatgttttag22080 catcttcatatgcggtgttaacccctagttctgtacagcatattctgttcaagtattttt22140 ttacaagcttgtgctgtaggcacatgccttctgctgcagaagtggacgcccgtggcacac22200 tccccccccccccccgtggggtgccacgccttcatgggacattgccacttctgccctgga22260 actcgtgcaggtacgtagtagctgctactgccacaacggcaacaccaagcaagagatggt22320 ccatgcttttctgacgttctcagaatagtggctagcttcaaacctgacaagcgctgcttg22380 aagccggaacactagagaatgttgctgagagcagaaacggccacgcgggtcacgactatg22440 cgtgggaaagtctcaagcttccctcctgccagcaacaagaaggctttggagtaggcatga22500 tgttttcacgtgtgcgtgccgtttctccaagcactgcaggttccaccgtgtgtcagaggc22560 tgcaagtttaacatcctcctgcctgaaaacaaataggtcctttgctgaaaagagggtaaa22620 aaaagagctttgatcttctcagccaggagaagagggtggtgttttcacgcgggcaactgc22680 tcgccggcctacatggggttaattcaagtctgctgcgagcacgactccgcccttggcact22740 ggcctccagcaagccctgttctctttggggtacaggggaacgggatggtttagactttcc22800 tgctcagtgtgtaaaaaatgtagctaaagccactatttttgctctccttaagctgttcaa22860 taaaccggttcctcattttacacgtgcatgatgtgtatcttctttgctggatgggccagg22920 aaactggagtggtcctctcagccagcctcagaggaaagaaatctctagctggcacaggca22980 gccagtgagtgaggctggcggctgcaggggcacagcctttagaatgagtccttcagtgca23040 caggtcccagggtatacggggtagtgggaggaaggaggggacgcctcgcagatgccactg23100 ttggctgggctacaccttgccacacttgttactgcttaggaggctttctggagtgttcct23160 tgggtgctacgacaatctgcagcagacactgtcctttcaccgctcctggtcctcgtttgc23220 tccccagtgatgtcaacagctgaggactgctcacgctgcaacaaaaggctctgcagtcgc23280 tgtctagcttgccctagtcgtctctagagttctgcctgaactgaaactcaagtggggttc23340 agctcatgacttgtggcaattgaccaggaaattcaccagttgctgtggctggaaggattt23400 tcagtcctgtgggttgtaaccagaggccacaggtggattctgccttaggctcatgagatt23460 tccgacttgctgttgaagaaaatgccttgtgaagtgacaacagtagctctgacccaactg23520 ccggtgcctcgctagttcctatacgtcccactggatcctcacagccccgggaagcaggtg23580 ctactactcttatccccgggaggagacagaggccgagagaggttaagtgacgtgcccaag23640 tcacacagctcggcagcggccgggttgagcatcagcagtctgtttgcagacccctcactg23700 tcaccccctgagccagtgcgccttgggccctgcggtcaggatgtctcaagcgtggaggca23760 tcaccggttcgtggcagtctctggaaggtcactgagctctgtgcccagaatcgagtcggg23820 ggagtctgtgcagaggtggccctgtgtgtggggacagtgtgtgacacagacactgctttg23880 gatggacacctctcccgtgacctcctagcatccaatcccaaaggaacaactgttgcagag23940 atggaccgctggacacaaacccacgtgcgtttctctggagacactggccaaggaaaacaa24000 aacatgctcgaaggccaacagctgcatgccccaccgcgatgtgaccgcagacacccgggg24060 tgtagaagggtctctgcctggtggggggacacgtgcaggccgaggagaggcaggaaggag24120 WO 00/46360 PCT/iJS00/02405 gctgcctccgactccccactggactgcatggcgacggcgtgtggtggggcagtcagctaa24180 gccatttgcctaaggggctgtcgggcatctgcgtgctggggaccgacagtgtgggtgtgt24240 taggaggatctgtatggagcacattgctgcctctggctaggacagggtggaaagggtggc24300 gtggctacagcctgacccatgggcaccgtcctaccctttgttctgtgcttccgagtgtca24360 gtcatgtgctggggtctgtgggcccatgactcagacggtgagctctgaccttcctgagcc24420 agggctttgctgtagttgtgcctggctcaggagctctaggacaaggggaccgctccaggt24480 ctgcatctacggtgtggcagggcccctcggcactcttgtgcactagtgtcatctttccca24540 ttgaaatgactgtgaggaccagaatgtgcacatgcagatgggcagctacttgtctgcctt24600 ggccctttattacacaacttgctgggggtggagatgccaccccccggcagtcagagcccc24660 tttatgatgtcatggggctggttacatgactgccaaggggtgctgctggccacactgcac24720 tagcaagtttgccagatggaggacaagcgatcattgagtatggctcgctgtgaagaaaga24780 aattcgagaggacaggatcatggcttggaaagggtgcctttccctccccagttgcagtca24840 gagacctaccttcacccagcagatccttcccctgcctgggacgacccggggtccactggg24900 agccctaacttgaggctgctgacagaagaaatcgctttccaacctctggccgaggaagct24960 tcgttcagaaggccgcaccctgacggtgacgtcccgccccagggagaagataatctcctc25020 tccctcccctttccacagaaactgtggagactggtcagcagcaaccagttttcgtccatc25080 tggtgggatgacagtggggcttgtagagtgatcaatcaaaaactctttgaaaaggagatt25140 ctcaaaagggacgtcgcacacaaagtgtttgccacaacttcgataaagagcttcttccgc25200 cagctaaacttgtatggcttccgaaaacggcgtcaatgcactttcaggaccttcacccgc25260 attttctccgcaaaaaggctggtctccatcttgaataaggtaatgaacgacaagcctctg25320 gaggggttaagtcggtgggctctggggcctggtcgggtggaagtcccaggactgcctcct25380 gggaagtgggcgacctcaggcagggtgtggggccatcgctgtgggcctgtgtccccctct25440 gggtggaggtgacatgaactaagagtgaatgtggggagagggctgaggatggtgcgggcc25500 cctctcgagtgtgtaaaatatcacaggtgccaagtagccgtatctgcgtgtcgtcctccc25560 cggggccagccatgtcatctggtggttgctgtgtccccctgactccacagcacattaccc25620 tgtgaggtgagcaggccaggggagtctggtatttgtaccactgtcaccctagctggtgtc25680 tggagaggtgctcaagtggaagcactgaagggcgcctggcgcaggaggtgcagatgctcc25740 tgctgcccttggtaggtgggcccctggtgtggaagagccagtacccagggcctccaaccc25800 agccggggtgcattctgttgccagctgacactgcatgggggaggcccagaatcttcttcc25860 ctcctggtctgcaacttcaaagaccctttccgccggccatggacaccctaatctgccatt25920 ttgaggctttttccaagacggaaaggcccgccacaacttggtaaaccttgacgatgtgaa25980 cgcgagtccccagcttcctttggggactgggaccttttccagaaaggcctcctgggccag26040 tagagttctcttgcacaggggcgtagatggttggtagttgtagtccatccttgtgacttg26100 cagtttccttttgtttatttaaactttgacttatagttagagttctactgccatccttac26160 tttcaaagagactcccctcacctcctcgtgaggatgaagagaagagtgggtgtcaagtct26220 gcaccaagacatcaggaggaggacaagccagaagctgctggatcctgtctggcaccagca26280 gacactgagcaacaagatcacacgtctccgaatgagaatgaccaggtcacaccgcaacac26340 cgggaaccggccggtcccaacacccaaatcaggagtggctctgctccaccagcaactcct26400 gtgatggtgcctgattccgccgtggcgagtgacaacagtccagtgacccagccggccggc26460 gagtggtcagagggcagccaggctcacgtcactccggtggccgctgtccctgggcctgca26520 gcgctgcccttcctctatgtccctggatctcccactcagatgaattcttacgggcctgtg26580 gtggcccttcccacagcgtcccgtagtacccttgccatggacaccacaggacttcctgca26640 cctggcatgctgcccttttgccatctctgggtaccggtgaccctagtggctgctggggct26700 gcacagcctgctgcctccatggtcatgttcccccatctcccagctctgcaccaccattgc26760 ccccacagccaccgcacgtcacagtacatgccagctagcgatgggccccaggcgtaccca26820 gactacgcagaccagagcacatagagggcagcatttgggcagaatatgtgctggtcaata26880 aatgtgtcagaaaatgagtaattttctgactgcacaaaaaagtcttcatggtctccatcc26940 tttttctttctttgcagtccagtacccccctccgacatctttggcttgcatgatagagac27000 ttcagcctaacacgcctccctggaaaagtgcccaggagatactcgagcaggtgctgggtt27060 ttaggaagcccctcagacatcccgcctctactcattaaaaattacctggagctggccggg27120 cgcagtggctcatgcctgtaatcccagcactttgggaggccgaggcgggtggatcacgag27180 ctcgggagatcgagactgtcctggctaacacggtgaaaccccgtctgtactaaaaataca27240 aaaaattagccgggcgtggtggcgggcacctgtagtcccagctactcgggaggctgaggc27300 aggagaatggcatgaacccgggaggcggacgtgcagcaagccgagatcgcgccactgcac27360 tccagcctgggcgacagagcaggactccgtctaaaaaaaaaaaaaaaaaaaaaaaattac27420 ctggagttgctcgctgagtgctaattgtgctgctagcattgccggcctagggttcacaat27480 gactctgtgttactggccattccttgtcatggcggtgcctttatataacaggatcttcac27540 tttacatacagatgagatcacgtctggctttgtcattgggttaaaaagtgcccttgatag27600 ttgtggaggtgacacatactaccgtcgactcgaatagatctgctaggcgccaagcttgtc27660 agccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggcaccagctc27720 aatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggactaaaaaat27780 gacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccag27840 aaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttcccacgttac27900 gtaacttcccattttaagaaaactacaattcccaacacatacaagttactccgccctaaa27960 acctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccaccccctcat28020 tatcatattggcttcaatccaaaataaggtatattattgatgatgttt 28068 <210> 17 <211> 30115 <212> DNA
<213> Artificial Sequence <220>
<223> Modified adenovirus <400>
aaacatcatcaataatataccttattttggattgaagccaatatgataatgagggggtgg 60 agtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaag 120 tgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgttt 180 ttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttg 240 tagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataaga 300 ggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgc 360 ggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcg 420 ttccgggtcaaagttggcgttttgattcggccgcttgggtcatagacttctttgagaacc 480 agttataagctatggtttctctccacagaaaaagcacttatggtgtctccccctttccag 540 cccaccaacattttacatctaatttgggggggttttctggaccacttaatacccatccat 600 ggatctcatgtgaagactcccctggcttgagaaatcactgtcttgttgaaaatgggaaca 660 aagctaagtcagatagctggttcatcagcaatgactttgaccaagcctgatcccacccta 720 ccccaccccaccccagtgaccaccccccacaatggagcacacaactctaaactggtttgt 780 aggtatgtgtgtgtgaacacgctgaggaatctgcaaaaccaaatggtgagtgcaaaacca 840 aacagtcacgagtaaatctcacaacaaccacgtcctgagctgcagcccttgttgaactat 900 accccactagggccccaagattttaggacttgtgtgtgggtgggacctcccctttctatc 960 atgctttagaagacagggatttcaccagaattgaacatattgaacatatgacccattttt 1020 ttcagccaaaggcaattaaaataacttcatacttgatatccatgtcagcaaaagctgcaa 1080 aacgcaaatgggtggctgctaagagccctggtaccctgacgagcacaccaagtgcttagc 1140 aacagtggtgtccaaaggaccagctggaagcctgccttgatgagaagttgctcttctttc 1200 tacatgaaggaacacctctactctcctgcttttaatacctgagctgtgagtgatcatcta 1260 tgtccattagcaaacatcccagtggagaaggaaacactcatacccgaaatctaagctaca 1320 tagttggaatcacttcaacttattgcaataaacacttactaagcacctattgtgggcaag 1380 tctttgcaatggataatagttcagtagatattttgatgtaatatttgaaataacaataaa 1440 aattgccaccactgaatttattgagcatttgctgtgctttaggcactaacccaggttctt 1500 taaatatttggtcttattcgatctgtataaatagccatctatgagaaagggactattatt 1560 gcccttattttacaaatgaggccaatgaggcccagagaggttaactaagttgcccaaaat 1620 catacagcccactagtggcagagcaggatgcaaacccaggcttgcctcgttcccaagccc 1680 acatgtcgtttgcattggtgtggaggtgtgcatgtgtttagtcattagcatgttatatga 1740 taagcaagttttgaaacatagaaacttaaaatgtgccattaagaaaagtacaggcaaggt 1800 tttccaaggggaggtgtggacctccggacaaatttttaagaactaattataaatacttaa 1860 aaatgggaataagaagacaacctaactacctgaacagttttagagatgactcatgcccac 1920 cctctaaaacccaaacaaaaacaacaaagtcaagaaaacccatgaaatcttagcaagcga1980 tttctatgtacttgtgaaaaggatttctttaccattctaatgggatttatgccaaccata2040 gagggctcagtgcccctcccatggggtggttagtgagtacagagctgagctcaccggcca2100 tctgcagcttcatgttatcaagctccagtttgtccttggagcaaggttatctgggacatg2160 agcagaggcattgctttctgcaatggacagttctttctgcctgcatacctagctccttga2220 taactttaaataccattttatagccacactggagttttgaagacctcaatatgcaaatat2280 tactcaggttctgattacttgtctgctccatgataacacactctaaaagcaatgaatggg2340 gcttatttgtagagaactgaagcattttaagcttttgctcaggaatccctggtagcttcc2400 tgtgacttgcaagatattagtgatgggtcaagaaacaggaccccccatcagcataacata2460 cgcagtgcctcagtagtccatcaggcagaaaaaactgcagatggcacatggaaatgacca2520 gcggcggaagataccccgacagtgtgggcagttctatttcagcagcaatcaagagggggc2580 ctggagccactcaatcaagtggagcaggatgggagcaagcactgtgcagaccaatgcaat2640 ccccagttaacacaaaaaataaataaaagagatgagattcagtctcttgactgtgactga2700 ctgggagctttatagctgatgcttgtgtcttttctccattttatttaattaggaaaagaa2760 atgcttatcacacactctacgtgtgaggtacaacctccacaggaaaggttgttaggaaca2820 tttcaacttctagaagtttctaaacataaggtaaatccatctttgtccttgggatcactg2880 cacatctcagaaaggcaaataaatcagtaattggtgggcataattactagctcatggact2940 gacaaggtctacactatttcgaatctcacagaagtaagccatgggacagatagagtctga3000 tagtggtgccccgtttcctggaggtcacacttactcatccccctggaccctgggcttctc3060 atgattgtcagagagtttgctggaaccaggtcagcccagtttcccttcccctgaaaaatc3120 ctccaatggctcgcaccaagactagagatgcaagtgcaagcacatccaccctctcagcag3180 ccaggttttgcgttccataatgtcacgtacccccagtcataccaatctccttggagctct3240 ccagacaggctgccatgtgtggtcggccctctgtgcttgtgctccttggtttgccaagcc3300 tggaatgccctttctccatgattgtactctgggaattctgtgtgtctttcgagatgaagc3360 tcctccaccctggaaattctgcctctctttccaggtccagctcctgtggctgaataccct3420 tggctgactgaacattcttgctgggccaagtcttaatcatctctaaatccctctggtgcc3480 ccgacagtatctagcccaggcaaagggctcagcaaatacttgcaaaattcaataaacttt3540 actatgttactagcttggcatgatgttctaggcacttgggagattataatctggtgagca3600 ttgtgctagtcttttttttgggataaacaccaatgataaactgagcctttactctcattt3660 tagcacatctactcatctcttttcaatgctggtgggtttataaaacccatcagtagaaca3720 gagagtgatcataatcatatggtgaaaataacatcagctaacatatttactgaacatgct3780 ttagtgtgctgggcactgaactctgtgcacatgtgaatttgagatagattgttcctagct3840 aataagatgaggaagtggagatgggcttactcaagtcacacaatagcaagatggggtaca3900 gacagaaaccaagctagaaaccaagcaacccctgggtttggaaatgcatgggctcctcct3960 ctgcacatggcgaggagcagtcaggtgctcctccttctctcatactgaaataactctgca4020 cttttggctattctgtgacactctgtgctattctgtagctcaaatggctctggtgcaagg4080 agctcagattatgactaacccccatagatattcagctgctttgcaagaagtggatgaatg4140 ctctggctttataaattattgactagattggatattggcccaatctccactctggtgatt4200 cggaaggaggcatatgcacatttgcaaggttatagtagtgcactctaattccactggctt4260 ctgagagcttgtaggtttcttgacttaatcattctggaattaaggtaatggatcctcaac4320 accttttctttccctggcctttactaaccatgtaacagaaatgagcagagaaaacccaag4380 aaagcgaactggagacttgatgagtgtgtcaaagatgctcagagtccaaggtcctctgtg4440 gctcactgacttcagaagccaacctccgttgttcaagtcacttgtgaggttactatgcta4500 gagcactaaatattatccagatagcccaaagaggtgaaggcagacatgtggaagaacctg4560 gatttttgggcaagttgatgaatgcctcctgccccatatcaaagagaggtgataggagac4620 aactttgtgaatttgaaataatgcaccggggaaacaggaaaacgtaatgtaagtcacgct4680 tcttggcttttttcttgccattaccctacttggccaagtgcaaatgggatttcaatatat4740 cataagtatgcatctattaataacaatgcaagaaagctgtacaactgaagtctgagattt4800 tgtaagaaacagaatctctgtaagcatcaccatccaacagaacttcctgagttgatgctg4860 aatcatcccagaaagaaggcgcgccagctctgcaggatcttcaagtctggggtgccacca4920 gcaagcgacggtcctccatgggctcttcaccttacggcagtgtccagaggcaccgccagt4980 cctctgctcctatgctggtcctgctgtccctggcaaaaggagccagagcattctctccag5040 gcctcccgaggaggctgcttcctttgttttgcagatggaggctcccatcctttgttctga5100 atcaatgtgctccaaagataagccccaagaaaacagttgttgccttttgacactgacaat5160 tagaatcgttggaaaatggagaaaacaggaaatggcaaatggtttcagtgaccaggagga5220 aaccgtgcctgaaagttgctgcttagtgactgggacactcgctttctgctctcttatgaa 5280 ggacagcctaggccgtgtggccttttataaacaaagctatgaaggggtcgtcaaattttc 5340 tagggctgcaactgtggcactacgtcctgttgtgccaggtgacactgacaagcagcactg 5400 agttctatgcaagcccaggtgtgcttctctcatggtgacccccagagaactaaggcccag 5460 ctcttcctctgtcacacccctcccagcccccactgtcagacaagggaccacattcacaga 5520 cagtctcagccaagatggcaaccttggaagtcctggggatgcctttctagaagctttagg 5580 ctgacgccagggagctgtaaagcccccacctgtccctgggggttgtgtgctgggcaggag 5640 agaggggaaagagcccaaactccaccactgcctgctggcacaactgagcccacgccaggc 5700 actgcaccagcttcactcaccagctaccacatgctaatgtctcctgtttacccaggtgca 5760 atcacctgagtgctgctttctcaccactgcatgggaagaacgatctcattaccaattcaa 5820 ccacttaagcaggcaaaaggactccaacgggggaaaggcagaggacaaacgattcgggga 5880 ctgagacagatgcccacaaaatgttcctggattttagccggattcgggctgaagttcctt 5940 gccctatgagcccttggaggagagcatcctgactcagacaagaattcaaatgacaagact 6000 tctgggggatggcagtgaatggatccaaagccttgttttataaagaatgtggaggaaggg 6060 aggaaggaagggagggaaggggagactgagagggagagagaataaagaagaggaagagaa 6120 ggaagaggaggaggaggaaggggaggggagaggaaaaaggggagggtagaggaggaggga 6180 gaaggggaagggaggacagaagaggggagggagcggggagattatctacccctccatgct 6240 agggaactcctgtctccttttccagcagacactgctccatgagtgcccccacagagagcc 6300 agcctgccacacagcagaaggtgctcagctatctgtcccaaaggttgagcactagtgtat 6360 accacaaggaaaaaaaccttcttttgtactgtttggaagccagaattagaaacaactcgt 6420 tcacccttctgactttccctttgcaatgacaggaagatagtatactgcatgggaccacca 6480 cacattcaagatctgagttgtggggacccccacccccacatgggtggagccccctgagac 6540 ccgtgggctctgactggccatggcccatagatagtgacaacatacacactgctatctgaa 6600 gcctagttggaggacggatggatgcttttaggaaaaaattccctttagtcgtccctcaga 6660 aactacaacggcttcctaaaatgaatggcaggcaagttttgattgtttcctaagttcccc 6720 ctaactctcagagcctgtgactcagcttctccagaatgcctgcagggaggcaggaccact 6780 gggagtaatgagcacgaattgggccacttctcccagtggactgttaacgccgagatctgg 6840 ggtggaacccactgaacacctgcccttttgcgttgcccagaatgcagccagagggatacg 6900 ggccacagtaactctggggcccctgaacctaccttcccaagaccctgacgggaaggtatt 6960 gcttacatgacagtccagtcagtccctctgtcactcagttaacaaacatggtcatgggtc 7020 tactatgggccaggctctgttccgggcatggggataacaccgtgtgtctctactcttatg 7080 gacacaagtaaatcaatcaaagcagataatgttctaatgaccacccagtagggtgaaggg 7140 atggagggtgggcaggtgctggcagaaacctaaggtcaagtcatgggaccccaatgggga 7200 actggaaactgccacaccattttctatgttaggggtctttccttgcccttctagagacct 7260 cacccatggtaatgatgctgccctcaggtttgaactggtctgaagctgtcactgggggct 7320 tcacaaaatgaatgccactgaaagcaaacggcagagatctgtatatgaaattctttttac 7380 cctttgttttctgtcttcccacatgatctatgactgctgcaaactctattctgactcctc 7440 ctcagtcttggccggtctgggaagaacttggtttagatgctgggaatgatcccgggcaaa 7500 ggaaacaaatatatttccagcttgcttttcttcttcccctgtggtgcctgcatcctctgt 7560 ctcgcactgactcacaggctggactgacaaagtggctgtgttgacagggaggatttagtg 7620 gatgccaggcctgaccctcaaaggccctcaggtggggccaatttccaaggttcttagaga 7680 agatttggggccacaaatctgctctgccacttcactgctgggtgaaagctgttggcttga 7740 gaagagagagaataggtgagggacaataattctcaaggcaagctagctgatatcctttca 7800 tttgtggcttccaaagacagggaaaggcaaggcagaccagggtggaaacagcgacatcct 7860 gaggggacgacgtccgttttgctaattctgtttccctggcagctttgtgctggacaagat 7920 ttctctggtttcagctgaagagtgaacagctgaggagctctgtgactctgagagagagag 7980 aaatcaatagcccttgaaaggaaagcactgcttgttcttccaggccagggcaggcaggat 8040 aaagagaacaaggctgtacataccaaagccgaatttccattaaaggccagaatcctggga 8100 ggccctttcagcttaatgaccatcttctgatcactatcacttagaaaatgttcatgtctc 8160 cagttttctctgcagatagtgcagacatcctgtgcacattgatattttatgcaccagatg 8220 caattctctgcaaaggtcccctctggctggtgtccatccgcttgctctcatgaccactga 8280 tgcgactctgggggcaagtcaagccattccttctagactctagaatctagaggatgatcc 8340 caactcacaggcctcttggacaagcttgctttggattccttctgcctgtactacccagac 8400 tgccagtcctccagcagggcaatccaacttcctgcctctagggtgtgggagtcctgggcg 8460 gggtttggaatccacagcagatggctggcaggagaggcagttcccgagcacccactaggt 8520 gccaagcaggcaggtatcaggtgcagggcttcggaggtgcagaagcacagcccctgtccc8580 cctcaggttcccagtctggcctcagggacagacaggcaggtaggtatggaaacggggtac8640 tggaaggcatacagctgagggagagagcagctgcaccctggtcaatcccccactcatttc8700 cctggaggtggggctgaggcacctggagcactctgcttgcttgggttgtccctttgccaa8760 agaatgccgggcagaaagtagagaacaagcaggcccaggaagcactagaagtccctcatg8820 ggtggacagagctttaccttcggagctgtcacctcctgtgttcctcccaacaaccctgtg8880 aggcaggcgggcaggctgagcaggggactgtgccccgttgacagatgaggaaagcgaggc8940 tcagggggaagtgacttacctgtggcgtcgcagctaggcagtagtggagcgggcacctgc9000 ctccaggccttcggatcccagctgccccagggcgctgcctctgaggtgctgtacgaggag9060 gtgactcgggccagctcatgagcaaaggagctgccgggcaggaaggactcactccccagg9120 gcctggcaggacgggggctgtcgcccgaggctgacctgggggtgcctggcttccccaagc9180 ttgcaggctttgtctacatgagtctacaacaaccgattgaacaatccatcagctgcttgg9240 ccacaaaatggttctgactgatcttgtcactgccctgagtgtctagctgggtcatctggg9300 tgttgtggttatacccaccaactgctttggtggaacgaaaccgcggccgatctgaattca9360 gatcggccgcgggatcattcctggactcagattgttctgaagaagcccagttctgggtgg9420 catcaagtgcttgctagatggggggcttgccttgatccggctacacttggaggtgacttg9480 ttcttggacggctacatacagaaagagagaagtggggatgagttccaaaggcatcctcga9540 cttcggctgtggccaccggagggtagctcctggcccaacacggacttctcacctcccgcc9600 cttggctctctactgagctcccccctgctccccaattcctcgccattcccctcatttctc9660 tgccctcagcctggactgcagttcttctgggaagctgccccaactccctaggtctgtgct9720 caccaagagcagatcacactggactgaaatgccagctgatttgtctcttcaagaaaattg9780 gaagctcctggaggtcagggtccatgtctgcttttacactcagtgctctgtatgcaggcc9840 tggcactgcccaccctttgacaggtggtgcatattttgtagaaggaaggaaggggccagg9900 tggggtgggctgggctggtggcgggagctagctcagcctcttagattctctacccgatgg9960 atgtgacctgggacagcaagtgagtgtggtgagtgagtgcagacggtgctttgttcccct10020 cttgtctcatagcctagatggcctctgagcccagatctggggctcagacaacatttgttc10080 aactgaacggtaatgggtttcctttctgaaggctgaaatctgggagctgacattctggac10140 tccctgagttctgaagagcctggggatggagagacacggagcagaagatggaaggtagag10200 tcccaggtgcctaagatggggaatacatctcccctcattgtcatgagagtccactctagc10260 tgatatctactgtggccaatatctaccggtacttttttggggtggacactgagtcatgca10320 gcagtcttatggtttacccaaggtcaggtaggggagacagtgcagtcagagcacaagccc10380 agtgtgtctgacccacccaagaatccatgctcgtatctacaaaaatgattttttctcttg10440 taatggtgcctaggttcttttattatcatggcatgtgtatgtttttcaactaggttacaa10500 tctggccttataaggttaacctcctggaggccaccagccttcctgaaacttgtctgtgct10560 gtccctgcaactggagtgtgcctgatgtggcactccagcctggacaagtgggacacagac10620 tccgctgttatcaggcccaaagatgtcttccataagaccagaagagcaatggtgtagagg10680 tgtcatgggctacaataaagatgctgacctcctgtctgagggcaagcagcctcttctggc10740 cctcagacaaatgctgagtgttcccaagactaccctcggcctggtccaatctcatcccac10800 tggtgcgtaagggttgctgaactcatgacttcttggctagcctgcaacctccacggagtg10860 ggaactacatcaggcattttgctaactgctgtatcctaggccaataaatgttgatcacat10920 ttatagctgccatggtagggtggggacccctgctatctatctgtggaggctctgggagcc10980 cctgacacaaactttctgaagcagagcctccccaaccccttttccattccctatacctga11040 cagatggcccaggaacccattagaaatggaaggtcactgcagcagtatgtgaatgtgcgt11100 gtgggagaagggcaggatcagagccctgggggtgtggcagcccccaagtgattctaatcc11160 agatcctagggttgtttccctgtcccattgaaatagctgctttaaggggcctgactcagg11220 gaaatcagtctcttgaattaagtggtgattttggagtcatttagaccaggccttcaattg11280 ggatcctgctcttagagttggatgaattatttaactgattttcagatctcctctttctca11340 atgctttcagaagcacagtaactgcttactctgaaatgaattctcaccccacttccacat11400 atgcaccccttgcccacccctttgggaacactggccttaactgcttaccttcaaatggac11460 tcatctgttgggagatatatgcattctgccgttcaggggtcattgccataagacctgatc11520 tctgttcctcttgctaaacagaagatgaaaaagacaaattagattacagctaccaattaa11580 taattagccttaggatcgctgcgtggggacctaggacttggctttggtgcagcagaaagc11640 atgaataaacacaccagcatacactcgcatgcatgccccaccctctcgagcaaaattcca11700 caggtataaataaagtaagattctgcacctgggttaaaaacacaactgcaacagcataga11760 atggggcaggagagacagaacttaatagcaagagcacacagaaaaaagttttaggcattt11820 tggatgtccatctgctcaggatgggtcagcagtgagatgcggtcaccaaaagaacaaatg 11880 taacattaggctgcattaatagaagcagagtatgtagaaggagggaggtgacagtcctat 11940 gctaactctgccttggccagactatacccacaggagtctgggcatgccagtctcagggag 12000 acccagacagactggctgcattcagaggatggtaagtaatgagagtggggattggacttc 12060 aaactacccagacaaagaatggctgagcaagccaaggatgctgtggctggggcagagcag 12120 actgtgggctatgtagtggtggatacctagcctctgcagggctgtcatagggaaaggaca 12180 ttgagaagaggactgaggcttgttcctggtggtcctggcatgaacggccagatgatcaca 12240 tggtcaggtggacacagtctccaacactgggagtagccaaacacttactgccaacctccc 12300 gcccttctcctgactagttgcagcataggcaattgggaggagcttcctgtctccatctga 12360 aagctggctgggtgggcagggggaggagcgagccaagtttcaaggccgcagtttcagcac 12420 tcagtctgggatcggctcaaggagcaaaggggaagaacatagccaggagggaataacatg 12480 aaggcccccagacccagaaaaggcatgacttgctctgagaccctcagccggttggtgtca 12540 ggttgtgactcggatccaggtctgactcccagtccagtgcttgaagcctcaccccacaca 12600 gtgaggggagcccggccatctctgctcaactgctgccatctctctccccttctcaaccac 12660 caaggcagctctgtctgggagcacaagctccaagtccactttctggtctgtgtccccccc 12720 aagatgccagaggacttgcctctacaacacgggctgcccgtgcagtgcctgcttttccag 12780 caaagggcttctgggaacccttctctgcactcagtggggctggtgggagtggggcggggt 12840 agcgacccagtgcttgggactgtgcccagctctcaggcctggcagcagttcctggccttg 12900 gttcctgccaaggcagagaggacaaacacatggcaccgggaagactacaccagaagcgat 12960 tccaccagactggggtttgcttttctatcccgcccttagcctgcttcctgtcctggtccc 13020 tgcctccccctccactggagctgccgtgtgggcagtgaggggctgtttctcagctgccct 13080 atggagctgccctctccctgccaaagcattggcaaggcggcaaggggtgggggtggggat 13140 ggggggtgggatctgccttctcaagctctcattatactgagcacgtctcacccattattt 13200 tatgtcatctagcaacaccccatgtggacactgaggagcatgggggtcacatgaccactg 13260 cccaaggccacaccatccggatctgcctgagatggtcagggttggcagccatttctgaag 13320 gcagtcctttcgctttggctcttcttgtaccagtctcaggacatcagggcagaagatcta 13380 cagtccccagcttactgatgtgacagcagaggctcagagaggttaaatgacttgcccaag 13440 gtgacacggctaagaagtacagtatctcctaactgcagaccaggtgcttctgctgcttct 13500 ggggacagattcctgcgtggctggctaggtctaaacggtccttaactccatccccaccgg 13560 ttgctgcattagtttcatcaaataacacagttgtacagaggtaggggttcaggggcaggg 13620 gcagatggaggctggagagtgtgactaaggaaacagcaggggaagtgcggtaaagtccga 13680 agggagggacggaaagagaaagccaagcccaggggcgtgccagacaaaaggaaaggccac 13740 gccggggcagggcaggcttcagcgggtgctggggcgtcttcatcccgggaagcacacatt 13800 ccagaggaccccggagtctaatggaaaagctggccagcctatcactatggaaactgccaa 13860 ggccacacagcgctacgcgtatttaaatgtgctggagtgttgagatactgtagtggtggg 13920 atgcttaagtgctggggtgctgggtgttgggatgcctaggtgctggggtgccgagatgct 13980 ggagtactagtgtgctgggatgctgaagtgctggggtcctgagatgctgcagtgctaggg 14040 tgctgagattctgggctgctggagtgttggggtgctgggatgctggagtgctgagatgct 14100 tggacaatggggtgctggaatactatggtgctggggtgctggggtattgagatgctaggg 14160 tactgggatgctgaagtgctgagatcctggagtgctgggctgctgggccacaggctcttg 14220 aatccattcgtctgcccaggggaagaaaccagaagataaagagctaatgaaggagctttg 14280 gttgagagggaggaagtaatggaaggagcaacatcttgtggaggagcaggagagaatgga 14340 cctcaggttgggagagagggccaggctacaggccagagaggcagaaggattccagcagag 14400 tgtgggctccaggagccaaggggaaacaggtttctgggaggagagagtccagtactgctg 14460 aagtggcaagtccgctgaggaccaggaagcttcatttggctttatgaccaggaggaattt 14520 ggaactgtgactagagtacttagggggaaggaggcaagactggagccagattgctctggg 14580 ttgaggggtgagtgggaggtgaagcagggcactgtcactcctttgaagggtggcagagag 14640 ctggaattggtgctggatgggctgtggggtgacagggtcatgtggaaagcccctgggggg 14700 cacctggaaaaggagaagctgacagtacagtgagaggacagctaagggaaagcggaatgg 14760 cagaacacgcactgccaggaggaatgaggatagggtcaggagtgccaggggcagtgaggc 14820 cagcctggggtcaggtggcaggacgtgtccaggaagctggtctgcactgcagcccacact 14880 ggctcagccttgaggttccctgtgtggttggggtaggaagttgaaccctctgggaatgga 14940 agatggaaccagctctgcgagccaagctcagcttttatctatgggtctctgagggctggc 15000 agagctgagtggggacaactgtgatccgtgaggctctcaggttgaggtggcccctccggg 15060 agggcttcattttcccagcgggtaggttctaagcagcagtggctgggcaggtgggtccaa 15120 cacagagccagagaagggtgaatgggcctcctggcaccccacccctgctgcccctgagct15180 cagtgatggagggggacagcacagctgagcccaagtgctttggtgtggccctgagggaaa15240 gctgcagcctgcctggggcctggcatggatgggacacttgaggcagagggacaatagtgg15300 gcgctgcagtgaggctggctcttggagaggtttcctgaggagtgctgcctgagacgggca15360 gggagaacagagacaaagttggtgacagggaatgaaagctgactgaaggactttacccag15420 acctatgaggatatctctctcagcaggaagcaggaggggactgtgtgaggactggccaag15480 agctggagtgttgggaaaatgactctttctccgacccctctgtcctagctctggcccctg15540 gactgcggaggtctgcttccacccccattggtcgatcgttgtcccttgtcacagccattg15600 agaattttggcagggagcatgttcttagagcatttttaggctctgcgggacataacagct15660 ctgcctcagagcacatgcctttctcagctcctgaaagccactgatcaaattggaacattt15720 tgtaccttagggatgaggatatcaactctcccagccacttagagggataaatgtgatgat15780 gcattcaattgtgactacatctgatcccaactgttgcttcagctgctctcctatagcaca15840 tggcgggaggcgtgcatcccagtagctacctccccacttttggggagatgtggttccatc15900 catgaaacctgggtacccgcctaccaggtcctggcctatcaggtggcagggtctggtcaa15960 agaagggcatgtgtggtcttcagcaagggagacaggacggtggtgcagagcgtctagacc16020 ctcagggcaagtctcccccacacctgctcccggggcagttgtctttgtgacctcccatcc16080 ccctctgtttcatcctctataaaatgaggggctgagccccaaaataacaggcttctttgc16140 catgatgcaaaactgctgaatctttctttctgacacacaaggcatcgagcagcctctgaa16200 agaaccaaagccactagcaggcttcctgacttgggtttgtaggtactgaatactcccttg16260 aaaaataaaaacatagaggcacttttctcctggctgtttattacagaacgaagaaaaaac16320 acactggcttgaaacagacgccagatttcaaatgtagaggtgaaatacgaggtggcaatt16380 aaaatgtgattacagaaagtctggacactgagaaaagtttacaggacagtgggtgtgggt16440 tttctataacagacacttaaatatacatgacgataattgcagatagaaaccatcaaagac16500 aaaccccaaatcaactaataatgtttacagatgttcccccccaaaccacagagccttaca16560 tcaaaacaaatactgaaaggctttaaaccaggaacagctcgccttaaccccacgagggtg16620 cacacaagctgggctttttctctcggtctgaatggtaaagggaggaggatactctagctc16680 ctccaggtggattgctgagacagggctcggctcacacactgtctctgcgcctctcccaaa16740 tctggagaactctcccagcctcctggtaaagtgtctctgtggggcacttaacgataaaac16800 agcttctgctgtaaagctcattaggaaagagctagcggagactgaaaggttcgcaaaaga16860 gattaagaatcacacaaggcaataggatttttagtgaacatagaaataaatggccaagtg16920 gttttctatttggcatttgtcaacttgcacaacaactcttggtcatatccacattgctca16980 ttgcattaaaaccataagcgactcagccacctagcttaacaaggtatcactggagcaaac17040 aacacggtctgcatatttgtaacattgtataataaacacaaaacaatgcatagtaaacac17100 aactctactgaaacaaaagccgtcgctttatttacaaagtcacaaaatgaagtataaata17160 cttctgtcattaatgtttaggaaaaccatttacaaaattttcaaatatgtacacgtagct17220 tgaaaaatcaccagctttccattttgtcacaggtagagagagggataagcatgggctgac17280 aacaccactcaaattgtaacgggagacaactgcgggtatggatcgacaccacttcctaga17340 gtgatgtcaccatgggggtttctatgggcatcctgctcagatttaaagtgccccagcatc17400 ctgggtgacttgcccagaattctgggctgtggcattttgagcagcagcatgctgttccaa17460 aatgtcgtcgatcagcctcaagttgcacacccagtcttcatctgggctcacacaggagcc17520 tttcaagagagcttcaatgaaatctacctcattgcagtcaggtgacgaaatcagatcatt17580 tagtgggggttggggctggcgcaaaaagtcggcaggtggcagctcagggggaatatccgt17640 tctgtcgaacggacctgggaactggctggcagcaacggcagaagcagcagcagcggtggc17700 agcagcagccacatagcttggtggctcgatgccctgtatggggctcaggggactaaagct17760 ggccataccctgctggaggaacttggtggtgtttgctacaggcaccgggccctgtaccgg17820 gctctgcctgaggctctggctgcccagcaggctgaagctggggttgttggccaggggcac17880 ttgtgttcccatcgcagcgggcacttgtgcctcccaatcagatggcctctgaaggcaggc17940 ctggccagaaggtgagtgctgctgaacgctattatccacttggctgaggggtgttttccc18000 cgaaactgctgtggtcacagctgctgccgctgtgacccatgcagcattgttgaacgcagt18060 gggcattcttggcacactaggccgtctgagctggtggggactcaaggactgggtgcccag18120 ggagctgggacagaacccaggcaggggcacttctggtggggtggccttggggctctgcat18180 atgctggcagacagagtcaagtctgcccaggggagtctggcctgagtgtgagaggatggg18240 acactgggggctggaggtgaaaattccttgccgcttccccagagttggtgagatcactcc18300 catgcccggcgcgcgcgtcgacgtgagcaagtggaatggagtggcagggaatgagtgagg18360 gagacaacagctcagtaggaccttgtgggctactggaaagactttggctttgactctcag18420 tgagataaggaggtatttgaaggagttttagcagagaaggaacaattctgtttattttta18480 ggagctctttatatatttaggaaattacttctttgttagtcatatattttggaatttgtt18540 gttccccgtttgtcatttctgtttctacattttgcttataatatttgtctatgtaaacct18600 taatatgtacttggttaaattgataaattctttgcataattaatagctagctattgttgg18660 cctacctataattaatttttgctttatctagtaataggcctgaattttcatttgggatcc18720 agccctctccctgacactatgcagggcctgggagactaaagcctgctggaaataactcca18780 gcttggccctgccaaacattacaacacctagcacctgctctccccaccatagtgactgat18840 ttgggcaggaatgagaccacagccactccaagagcccagctgttg~ctaccagtaacatc18900 ttctcctctaaacaggagatccagctatattgaaggcttccaaaattaccctatgtctac18960 ctgtcacaggaatgttcagaggactaagcctctgcctttctccaccccatacttacttca19020 tatggaactatgaatgggcaacttcacaatttattatttacgtcacctagatgcaagtca19080 gagggagaagcaacaaatcagcagtacctaagaagttctcaagtcctcaaggtgaacttt19140 acctatatatacaaaatatatttgcctctttcctttaaccctttttcttactcccaccac19200 tttccacccaatgtgtaataatattagtcacttcatagagttcttatgagaagttaaatg19260 aggaaatcatgtaaaccactgagggctggcaacaagttcttaataagtgctattgagtta19320 tgagtattattatctcccttgggctggattcacagtgtcacctcttctgggaccatagta19380 agccaatggttccagctccttctattatgtcattttaatttggggaatagcttaacatat19440 tagatgatcctttacatgcctgagcggtctttctttgatggtatttctatgatcttatga19500 agagctagatgttgccgctccattttatcttgctggtggcagctggtgtttagccaactt19560 gtctttttttttctgtctttatatttttggttttgttttttggcagaagcctgtgttttt19620 cagattactttttgcttgtgtctactttttcccaaaacaatattacatattaaacttatt19680 gtatatttaagtttattttatgcatcaaaagtcgcattgaatttctgtgctattgtgggg19740 caaattgacatcaatgtaatttaactcattttatcccaaattaggctatgtctttccata19800 tactcaaatcttcttttatgtccctcaacagtttccttcatataaatattgcatatttct19860 aattaagtttatttttctaatctccattttggatgtcttaaatatagtcttttctccctt19920 tgctctttatcaaagatctgcaaactatggcctgtttttgtaaataaagttttgtaataa19980 agtttaaatggccactcccatttgtttacatgttgagtatggctgctttcagactgaaat20040 ggcgaatgaagtggtcataacaagaccatatggcctggaaagcaaatatatttattatct20100 ggcaatttacaaaaaaagtttgccaacctctgctttatatttttgatctagttgttattt20160 gaaaataaagctattgatatttgtatattaattttttacccagctaccttattgttttta20220 ttacttaatagtttacagtatttcttttagtttttccaggtgtaacatctgtaaatatta20280 ataaatctattagctaatatttcatttagcatttttatatctatactcacatgagaagtt20340 ttctttaattttcttttgtgtttatcaacctgtgttcactcaggaaaatgaagccactta20400 agtataccaggagtggagggtttcatatagaaatgagggcttatttgaattttgggaaag20460 ctgatgaaacaggtcagcagagttactgcagaagaccaggaaagttacatctggaaggtt20520 agggaagcagacactaaagacttaagcctgaagcatagaagtaaggatccgtcaatactt20580 attgagaaacttctggaattctcaagaacctctgagaaatctcccattctgtggcaataa20640 agtggtggtactcaagccatcaccaacatggctcatatgtctcattgcactatgtccaaa20700 gacaaatggcctctgcttctcttctgtctcctaaatctcttaagatttcctcttatttgt20760 ttaagtctaacccagaagacttacatggcaggtaattctgggaaatgtattcccttgttt20820 ctccaaaggggtggggataataccaagttgacaacagaaaatctagcaccctttttaagg20880 ttttggcatcagtgatttcttggtgctctaaatagttcttatgattattccatgggtgct20940 taaaaagaatatgtattctctgatttaagaatatgtaattgatttaactattgattagat21000 attaataaaatagatttttaaattaccttattaatttggttaagtcctccatgactactt21060 attttttgactatttgttatggaaaggtgtagctaaataaatatagaactactctatatg21120 tgtttctagttctctatgtattctctacattttgctttataattgttttctttcttagag21180 acaggggtcttgctgtgttgcccaggccagagtgcagtggcatgatcataactcactgta21240 gccttaaactcctgggctcaagcaatcctcctgcctcagcttctcaagtagatgggacta21300 cagaagtgcatcaccacaccctgctaatttttttcttatttttatttttagtgatggagt21360 catgctatgatgctcagagtggtcttgagctcctggcctcaagcaatcctcccacctcag21420 ccccctgagtagctgggattataggtgagaggcaccatgcctggatttgctttataaatt21480 ttgatatgttacttggtgcatagatattcattgctgttagaccttcattatgtgttgtac21540 cctgcccttctttggttcacttttccttgaattcagtcttgtctgatattaattttcagc21600 tgtctgctttcttcttatttccatttttctaatatgcttttgcctactcatttgctttca21660 attttttctgagtcatttcattctagatgagtatattacagtcagtacttaattgggttt21720 ttctttgtcactgaatatgagtgtctcttttcctgtgtattgatagggtagatatgtgta21780 gtatgagtgttacaattttttaaaaaactttgatttctctcctttgttttatttggggtc21840 tccggttattttttaaggtgtatagtgtcattttatttttaatgtttgcctctgtaaaaa21900 taattttataggctgtacttaatcttctatttatttagctagtttatattgactcttcaa21960 taacagtagtaaatgtccctacttctaactccccttgccctccaccttttaattttgtgg22020 ggtatactactggccttagtttttctagtggttaattttatacatttaaataggtttata22080 ccttttaaacttcaatggccacctttaaaaatattttgaccccacccactataaaaaata22140 aacatgccatcttacacgcacaatctcactttctctccccttacattccctaatttttac22200 tgggtattttaatatttcttcatcatcgtggttgattacatttacatgtatattgcagtg22260 gtagttcccataattgttataacctttaaccatagttctacatttatttttgccaccgtt22320 tttcccattcaccttttggttcaactttatcttctagtagtgtcttcaaaaaagtgttct22380 tggaaactacagcccatgaaacattattgcatatttgaaaatatttatctgtcaccatta22440 cacctgaatgacaatttagctagctcaaatttactggctcacatcatcttttttttggta22500 attttttaagtacttctctactgtcatgtaatgttgtatgtaggatagcctaaagccaac22560 ttgtgtttttttattttgctgttatagtagtttgatcatttttgcctagatgccccatag22620 gattatttttctttgtctttatttcaggaaaccttactagactatggttcagtgctgact22680 agacatttttttccttaagaaacaccatgccctttagatccttagattcaaatctttttt22740 aatatcagtaaagtttttaaatttacatatttggatattttatacttctctagtttttct22800 tcttaaagaagaaaacattaattctgtctttgcctgtcttccatatctacaattttcttc22860 taatcactttttataatttccatttcattttgctcaattgcctcaaagctgctatctatt22920 tttctgttgtcaacaatctgtactcttacatcagctactatgataatattgtgtcaccat22980 ttactactatttacaagctaaccataatttctctgatgattttagttttctcttctactt23040 tgtgtcctagatacaggcagctcaattttaatttttgttgcttttcttgattccttatac23100 ctctgcattaagctcttaattcacaaattacttccattgttaagttattgagttcttaaa23160 caaatatttggtcatgattttcatctactccacatcaaaatgttcatactgtgtaggctg23220 tttgccttttttgtcctgctattttccaccattttgtgacatttttgtatggatctcctt23280 gttcgttttttattattagtcatccttgagtgagcagaaatcttcctagagcaggtattt23340 gaaagaagtagtgtggaagaggggctggggctgtgttctgcgttagtagaaaatccccta23400 attcttagaaaaatcctttatgattcatggtcttgtgggagaaggttgtgaagcgggttt23460 tgtgaatgtgcttcatgtgcaatagagagtgactgagaggtggtttaacctttcttctta23520 gctcacaaagaatggctgctacagagctgcttaacacccacagtttttcttctacatgct23580 gcacatttacaaagatggagtatccgtgagttttctcactcattcctacctcccctgcca23640 ttttaaagtatccaaggaagttggggagattccctcagctaaacagccacccagatctta23700 ttgttccaaacaaaaaattattagatttgccctctgagcaggtcacctactttggagaat23760 tttgtgctctgccagctctcctcaagatttggaaaagcagttattttctattggagcccc23820 catcactccagtccaaagttcactgcatgtgacatcccatcttatttgtctggttcctga23880 ttataggagtttcccagtttcaacaaaaatactgtctttgtttttgtttctcattcattt23940 ttccccaattttgtttcaaagcgaaaagtaaaaacattcttactctaacatcttaaaatt24000 aatgtctgggaggaacctaaactgctcattaatatatgccatgctatatgtgatatggca24060 gagactgctacctgtccaccagaatctatttatttttcttggatgcacaataagactaca24120 tttcctagtctcccttgcaattacatgtgaccatgtcattaagatttagtggatgaaatt24180 taagcagaagcaaggtgtgtaaattctagggctggcccattacaaactttaacatgggca24240 cctgtgtgtttttccttttctgcttaattgggttggagataatcacagtgatgctgaaag24300 ccatgtgctaaagaaggcagagcctccttctgcttgggctcctgaatgactgtgtggaac24360 agaggcccttgccactcttgggccattctggattattacatgcacaaagagtaaagttct24420 tttctgttaagccattagatgtatgggtttatttgctgttacagcctagtcgaacccaaa24480 tgatgtagtgagggaggcaggacaaggaagaaacctattcagtatggagttatccttagg24540 gacaagttacaaaaattataaggatcagacaccactgctggttgatccattatagaccag24600 aataagcaggtggtatgattccagaaggagatgccatggttgtgtgagagagagattaac24660 cattcctataaacagaagggtcaaagaaagtagccagtcaccagaatgcttatattaagg24720 aggattgtgactacctgtatataaagtataggaagaaaatggggcagggcaattaagatg24780 aattgcagacacaactatttgtgcatattaatttgaatttaaatttaaacttaatttaag24840 gataatttaaaggaaaatcttcccttacctatttcatgctgaaggggtctttcaagttgt24900 caaactctctctatataaataagactgcataatccagaccctccggaatccagagatgaa24960 ttgcatgtggtagctgatgtgtgggaagcccacagagcagcacttctggtcattgccatc25020 cagctagctggtgctctcagaagaatgagatgaagaggcatgactttcaagcttcctctg25080 ccatacaaggtgagtttgggttccaggcttcttcctagcccctgtttctgcttcctctga25140 tttaatacccagtcactgagtacctactaaacactcggcattaaactgaaaagaacaggg25200 aagtcaaaaaccttttaccaatgcctgatgggataaagacatgacccatccctcaaagag25260 cttgtggtcttcccatggcaatgcccttcctccctaggccagatggactgtgaagaacag25320 cagtctcaagtggatttattgggaagctcatgaagactcagctcaatgggccatgtttgc25380 aggggccccttacaaggctctgcacttgattttgtaattttgaattatttttctcaaaaa25440 ggcctcccaaattatgtaagcttcaggcctttaaaacttggatctgttcccttgaagcca25500 cataatctgatcagaaataattttcagggctcattgggtgctggagaattcatggttaga25560 tatttaaataaaaatgcagagaatctataggactttccaactgagaccaaggcttttttt25620 ttctggtagcttgactgccaggaaacagaaagcggggcatctccacctgctggcagaaga25680 ccaaaactacaccatattgcattgaccgcagacatcaggctttgtttccttttaagtgaa25740 taggtgaaatcaatcaagcaaactggaattaggaaacagaaggaccactcacaaccgtac25800 ctccacagcaaagctaacactttcattttgaataacccctttccagcccctgtttacatc25860 agtcacattttttgcatggacataatagtgtttcaggcagtcctgctctacagaaggcat25920 ttgtttcgctgaccataagcatcttatgtgcttctacattgtctttatagccagctgtct25980 gaagttctacatcatccgtcagtgatgggtcctgagtaaactcagcagtctcctctaagt26040 aacttgtatatatcccttttataaagaatgctgcagtggccacccttaagtatagaactt26100 tgtctttcccaggcattacttacaggaaactggggaccaaatgccactccatagcatagc26160 ttaggagaagtgagattccaaaggacagagtgggctggcacatatgtagtagaagactgg26220 acaaatgagagtgaatgagcgagtctgttgtgagtggatgacacgcaatgcaaccccaag26280 ggccggctccaatttgcacagtccctaaaacatcccagggatccagtcttatcgcagtct26340 catcaacattgacaataatcatttagagaattttcaccatgagacttttgacactctgct26400 ctgggattttggagttccaactgctttaacaatttatacagtcactcaaatgtagaatcc26460 agtgaattattaatgacaattttgctccgcatattgaaagggcattctatattaatcaat26520 tataaatcatgataaattgctttctaaaaattatacacttgaaacccagaaatgttcaag26580 cccctcaccagtttacatacatccttaaccgtgtcttgttaactgggtgaccaggaaaca26640 gagaaaggaggatgtacttaatgaataactggccagaagtcggaagacctagattttctt26700 cccatcccttccatagatttgctctgtgaccctagacaattcatatgccccctctgttac26760 catacttttaagctgaaggggttatagtgcatactttctgaggtccttctaacactggag26820 atctatgagttcataagatcacatagagtcttggacttttcagagatcactaggcataag26880 aaagccctcgaggacatcctcttcctagatcaaccactccaagctaagaccttaatgggt26940 ctacactgtcatccatttatataggagagaggatggtacaagctaagtgaaagtctgttg27000 tcaactcaggaactagggtacagttccagcaatcaatctccatggagggttagcagaggt27060 ctggcctagccaattagggtttggcactgccatatgctactagaaaacacatgctttcat27120 gccaggcaacagggtgggaagtgcaatgccattaaggaccataccttagcctacttaaac27180 tagcctgggattgagttactgagctcaaaatacaattcagcacactttcctgcccactgc27240 aagtcaggccagcccctggagaactccagggacaaggcaagtctggagtttgtccccaag27300 gaactcccagtctagtggaggagatgggcccatagcagagttacaaatgtccaaaataag27360 agcacaaattcctgggttcttaaagagagttcaagcacacacacacagagagagagagag27420 agagagagagagagagaaattaatttagataaggaagggaggtagggagagatggtgcca27480 gcaatggcttcctgaggaaatgtcacttgtgatgggtcttttaggtgagttggacattga27540 gagaaagtaatcaaaagggcacaagatagggtagctgtcaggtaaacaaaacaaaactgt27600 atgcacaaaggtctagaggcaagaaagcatgggtgtgcttgcatagcaatgaagaatgtg27660 gtttggccaaagcataggattcacagaaggacctccccaggccatcagtagtctctcctt27720 cgcaggagtccactgtggctggacgaggaacagatcaaaacaggtcttgtttgaggcttg27780 gcccaacaacagggccagctgttgaccagggcttccttcacccttagctttctctccccc27840 tagtgtagtcagagactccaaaggcagtcccagagtgttgtgccaaaacaaatgcacatc27900 aggctattgtcatcaggagcccttgtcttgttctgtttgttttaagaagacataagtgaa27960 ataaccaggtttccgaagagagggcagaattgggtttgtccccagggaaaatgaggtttc28020 tccaccatggctgttctccccatgcagcagcggccaggggtccccagcagctctttgctc28080 agtgcccctgatgctaggcctgatgcttcctatgtggtcagctcagatgtgcaacccttc28140 tccatttttattatttgagcaatataatcattattgcttttgttacccaaaatcttataa28200 tcacccatttaagagtaatgtaggaatcatttttctcttttatttgatgaactaatcttt28260 ctggttggctcttgaggtgggaggggtgggcaatggggaccagcctgcttcatctcactc28320 tttcctaggcctgctccttaggcacgtgccccacaaatcccacctccgcaggttggaaaa28380 ggggcccacagaggatctcaggtatctgaggaagggccagaggctatttggcctgacatc28440 ctggaacatcaccagccaggagccataactcctttaggctagcctgcctgcaactggatc28500 aggtatagcaaaaacgattgatctcaccttctaattttcaatgagtgggagtggctgcct28560 tgagtgctgtgttgggaaggagtctgaggcagcatctggaccctgtgtaaatatgcgttt28620 cctgttattgatgaagctttcaagacaagattttagaaagctttggctcaagttgtaggt28680 tgtgggaggaaggaattagtaagagccatcataaactcaacatctattgtgttccaggag28740 ctgtgaccactcccttctttttagtcgggaaggtcaggatctcactatgtcacctaggct28800 agagtgcaatggtgtgattattgctcattgtagtcttgaatttgaattcctgggctcaag28860 taatcctcctgcctcagccttctgagtaggtgggactactggtgtgtgccaccacactcg28920 gcttactcctccctttgtatcctgctgcatcgttagtgcatgatacccaccaccccctca28980 gaggcccagcctagtgctgtctctcagaaaaacaccctttgagtcaacagagacaaactt29040 agggggtgcctgactccaaagtccaggccttcctcctttgaggaacagcatggcttcccc29100 agggaatatcattccatggagacaaggcctgcagacagatctcattttcatctgttcttt29160 tactagcccttggaggatttgctccaaaaacctgaggcactgatgtttactccagcttta29220 cttcccccatacctggtgaacaactctggcccagcttccacacaccaaggcccatggctc29280 taatagagccagagaggcagtctcagctaagaagagctgtatcctccagggaccagcagg29340 aacttccagagatatcctacaagctccaactgctgggaaaagcagactggggtcttcctc29400 aggctccaccaaagatagccaagtggcagcaatgacatgagcacctcattcctgtcctag29460 accctggcaattccatgagagggaggctctgaattcacctctgttcccaggatgtgtgac29520 atcaatgcacattgtcataccagatacacctgccaaacttcagaacccctgggaagtgac29580 cagaatgccactgaattcaacaccttagaccaatgaacatgacctgggtcctgtaggata29640 ggaggcagcagcagtgtatacccagacctggtcgactcgaatagatctgctaggcgccaa29700 gcttgtcagccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggca29760 ccagctcaatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggact29820 aaaaaatgacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaaccta29880 cgcccagaaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttccc29940 acgttacgtaacttcccattttaagaaaactacaattcccaacacatacaagttactccg30000 ccctaaaacctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccacc30060 ccctcattatcatattggcttcaatccaaaataaggtatattattgatgatgttt 30115
Claims (41)
1. A nucleic acid molecule comprising a low homology packaging signal cassette flanked by a recombinase recognition sequence, wherein said packaging signal cassette comprises a modified adenovirus packaging signal, provided that said modified packaging signal has low homology to a wild-type adenovirus packaging signal.
2. The nucleic acid of claim 1, wherein said recombinase recognition sequence is loxP.
3. The nucleic acid of claim 1, wherein said recombinase recognition sequence is frt.
4. The nucleic acid of any one of claims 1-3, wherein said modified packaging signal is less efficient than said wild-type packaging signal.
5. The nucleic acid of claim 4, wherein said wild-type packaging signal is human adenovirus serotype 5 packaging signal.
6. The nucleic acid of claims 5, wherein the modified packaging signal comprises at a maximum, 23 bp of contiguous sequence homology with said wild-type packaging signal.
7. The nucleic acid of claim 5, wherein said modified packaging signal is about 2-3 times less efficient than said wild-type signal.
8. The nucleic acid of claim 6, wherein said modified packaging signal comprises two to six A elements, each A element having a consensus sequence of ATTTGN8GC.
9. The nucleic acid of claim 6, wherein said nucleic acid is a plasmid.
10. The nucleic acid of claim 6, wherein said nucleic acid is a helper virus.
11. The nucleic acid of claim 10, wherein said helper virus does not contain an E1 gene.
12. The nucleic acid of claim 11, wherein said helper virus comprises an E3 region with an insert of about 2.9 kb.
13. The nucleic acid of claim 12, wherein said insert does not contain a promoter sequence.
14. A nucleic acid comprising an adenovirus E3 gene having an insertion of at least about 2.7 kb, provided that said insertion does not contain a promoter sequence.
15. The nucleic acid of claim 14, wherein said insertion is a human intron sequence.
16. An adenoviral helper virus for production of helper dependent vectors comprising:
(a) an adenovirus genome having an E1 region deletion;
(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5'loxP
site, a modified packaging signal and a 3' loxP site, wherein said modified packaging signal has low homology to and is less efficient than the wild-type packaging signal;
and (c) an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.
(a) an adenovirus genome having an E1 region deletion;
(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5'loxP
site, a modified packaging signal and a 3' loxP site, wherein said modified packaging signal has low homology to and is less efficient than the wild-type packaging signal;
and (c) an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.
17. The virus of claim 16 wherein said adenovirus genome is human Adenovirus type 5.
18. The virus of claim 17 wherein said modified packaging signal comprises at a maximum, 23 bp of contiguous sequence homology with said wild-type packaging signal.
19. The virus of claim 18, wherein said modified packaging signal is about 2-3 times less efficient than said wild-type signal.
20. The virus of claim 19, wherein said modified packaging signal comprises two to six A elements, each A element having a consensus sequence of ATTTGN8GC.
21. A vector comprising the adenovirus of claim 16.
22. A cell line expressing E1 and infected with the helper virus of claim 16.
23. The cell line of claim 22, wherein said cell line further expresses cre recombinase.
24. The cell line of claim 23, wherein said cell line is 293 cre cells.
25. A helper-dependent adenovirus vector comprising:
a) a 5' ITR;
b) a packaging signal;
c) at least one heterologous expression cassette;
d) a human genomic stuffer DNA;
e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR;
wherein the overall size of the vector is between about 28 kb and 36 kb, and wherein the only adenoviral sequences present are the ITRs, optional E4 non-coding segment and the packaging signal, and wherein no bacterial origin of replication or bacterial marker genes are present.
a) a 5' ITR;
b) a packaging signal;
c) at least one heterologous expression cassette;
d) a human genomic stuffer DNA;
e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR;
wherein the overall size of the vector is between about 28 kb and 36 kb, and wherein the only adenoviral sequences present are the ITRs, optional E4 non-coding segment and the packaging signal, and wherein no bacterial origin of replication or bacterial marker genes are present.
26. The helper dependent adenovirus vector of claim 25, wherein said optional E4 element is present.
27. A plasmid vector comprising a) a 5' ITR;
b) a packaging signal;
c) at least one heterologous expression cassette;
d) a human genomic stuffer DNA;
e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR.
b) a packaging signal;
c) at least one heterologous expression cassette;
d) a human genomic stuffer DNA;
e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR.
28. A helper-dependent adenovirus comprising in a 5' to 3' direction:
a) a 5' ITR, b) a packaging signal cassette directly joined to the 3' of said 5' ITR, c) a first stuffer DNA at least about 1 kb, d) at least one heterologous expression cassett, e) a second stuffer DNA at least about 1 kb, f) an optionally present non-coding E4 segment at least 400 by in length; and g) a 3' ITR, wherein said 3' ITR is directly joined to the 5' end of said non-coding E4 segment if present;
provided that said helper-dependent adenoviral vector does not encode one or more proteins needed for viral generation, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.
a) a 5' ITR, b) a packaging signal cassette directly joined to the 3' of said 5' ITR, c) a first stuffer DNA at least about 1 kb, d) at least one heterologous expression cassett, e) a second stuffer DNA at least about 1 kb, f) an optionally present non-coding E4 segment at least 400 by in length; and g) a 3' ITR, wherein said 3' ITR is directly joined to the 5' end of said non-coding E4 segment if present;
provided that said helper-dependent adenoviral vector does not encode one or more proteins needed for viral generation, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.
29. The helper-dependent adenoviral vector of claim 28, wherein said virus is between 30 and 36 kb in length.
30. The helper-dependent adenoviral vector of claim 29, wherein said first stuffer and said second stuffer are derived from inverted mammalian non-gene or intron sequences.
31. The helper-dependent adenoviral vector of claim 30, wherein said virus does not encode for any adenovirus proteins.
32. The helper-dependent adenoviral vector of any one of claims 28-31, wherein said optionally present non-coding E4 region is present.
33. The helper dependent virus of claim 32, wherein said GC content is between 52% to 57%.
34. A method of generating helper-dependent adenoviral gene vectors in a cell line expressing E1 and cre recombinase comprising:
a) infecting said cell line with a helper-dependent vector comprising: a 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present;
b) infecting the cell line with a helper virus comprising: an adenovirus genome having an E1 region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.
a) infecting said cell line with a helper-dependent vector comprising: a 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present;
b) infecting the cell line with a helper virus comprising: an adenovirus genome having an E1 region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.
35. A method of generating a helper-dependent adenoviral vector comprising:
a) producing a cell comprising (i) trans functions needed for adenovirus generation; and (ii) said helper-dependent adenoviral vector, wherein said helper-dependent adenoviral vector comprises the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, and said helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%, and b) generating said helper-dependent adenoviral vector.
a) producing a cell comprising (i) trans functions needed for adenovirus generation; and (ii) said helper-dependent adenoviral vector, wherein said helper-dependent adenoviral vector comprises the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, and said helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%, and b) generating said helper-dependent adenoviral vector.
36. The method of claim 35, wherein late proteins and either E2 proteins or E4 proteins, or both E2 proteins and E4 proteins are supplied by a helper virus present in said cell.
37. The method of claim 36, wherein said helper virus comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence, wherein said packaging cassette signal comprises a modified adenovirus packaging signal having low homology to a wild-type adenovirus packaging signal.
38. The method of claim 37, wherein said recombinase recognition sequence is loxP and said cell expresses Cre recombinase.
39. The nucleic acid of claim 37, wherein said recombinase recognition sequence is frt and said cell expresses Flp recombinase.
40. The method of claim 37, wherein said modified packaging signal is less efficient than said wild-type signal.
41. The method of any one of claims 36-40, wherein said wild-type packaging signal is from human adenovirus serotype 5.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US11860199P | 1999-02-04 | 1999-02-04 | |
| US60/118,601 | 1999-02-04 | ||
| US13813499P | 1999-06-08 | 1999-06-08 | |
| US60/138,134 | 1999-06-08 | ||
| PCT/US2000/002405 WO2000046360A1 (en) | 1999-02-04 | 2000-01-31 | Improved helper dependent vector system for gene therapy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2360796A1 true CA2360796A1 (en) | 2000-08-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002360796A Abandoned CA2360796A1 (en) | 1999-02-04 | 2000-01-31 | Improved helper dependent vector system for gene therapy |
Country Status (4)
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| EP (1) | EP1151091A4 (en) |
| JP (1) | JP2002535986A (en) |
| CA (1) | CA2360796A1 (en) |
| WO (1) | WO2000046360A1 (en) |
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|---|---|---|---|---|
| US20020081707A1 (en) * | 2000-06-29 | 2002-06-27 | Donna Armentano | Methods for helper-dependent adenoviral vector production |
| US7820441B2 (en) | 2000-09-25 | 2010-10-26 | The Regents Of The University Of Michigan | Production of viral vectors |
| US9388427B2 (en) | 2002-12-02 | 2016-07-12 | Biovec, Llc | In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors |
| US7803365B2 (en) * | 2002-12-02 | 2010-09-28 | Biovec, Llc | Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases |
| ES2292271B1 (en) * | 2004-05-20 | 2009-02-16 | Proyecto De Biomedicina Cima, S.L. | AN ADENOVIRUS-ALFAVIRUS HYBRID VECTOR FOR THE EFFECTIVE ADMINISTRATION AND EXPRESSION OF THERAPEUTIC GENES IN TUMOR CELLS. |
| EP2020436A1 (en) * | 2006-04-28 | 2009-02-04 | Universitat Autonoma de Barcelona | Method for producing adenovirus vectors for gene therapy and dna sequences used therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6080569A (en) * | 1993-06-24 | 2000-06-27 | Merck & Co., Inc. | Adenovirus vectors generated from helper viruses and helper-dependent vectors |
| US5919676A (en) * | 1993-06-24 | 1999-07-06 | Advec, Inc. | Adenoviral vector system comprising Cre-loxP recombination |
| WO1997032481A1 (en) * | 1996-03-07 | 1997-09-12 | The Regents Of The University Of California | Helper-free, totally defective adenovirus for gene therapy |
| JP2002514904A (en) * | 1996-06-20 | 2002-05-21 | メルク エンド カンパニー インコーポレーテッド | Gene therapy for obesity |
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2000
- 2000-01-31 JP JP2000597420A patent/JP2002535986A/en not_active Withdrawn
- 2000-01-31 CA CA002360796A patent/CA2360796A1/en not_active Abandoned
- 2000-01-31 EP EP00910029A patent/EP1151091A4/en not_active Withdrawn
- 2000-01-31 WO PCT/US2000/002405 patent/WO2000046360A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000046360A1 (en) | 2000-08-10 |
| JP2002535986A (en) | 2002-10-29 |
| EP1151091A4 (en) | 2002-09-04 |
| EP1151091A1 (en) | 2001-11-07 |
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