Method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

FIELD OF THE INVENTION 
The present invention relates to a process of targeting the integration of 
a desired exogenous DNA to a specific location within the genome of a 
mammalian cell. More specifically, the invention describes a novel method 
for identifying a transcriptionally active target site ("hot spot") in the 
mammalian genome, and inserting a desired DNA at this site via homologous 
recombination. The invention also optionally provides the ability for gene 
amplification of the desired DNA at this location by co-integrating an 
amplifiable selectable marker, e.g., DHFR, in combination with the 
exogenous DNA. The invention additionally describes the construction of 
novel vectors suitable for accomplishing the above, and further provides 
mammalian cell lines produced by such methods which contain a desired 
exogenous DNA integrated at a target hot spot. 
BACKGROUND 
Technology for expressing recombinant proteins in both prokaryotic and 
eukaryotic organisms is well established. Mammalian cells offer 
significant advantages over bacteria or yeast for protein production, 
resulting from their ability to correctly assemble, glycosylate and 
post-translationally modify recombinantly expressed proteins. After 
transfection into the host cells, recombinant expression constructs can be 
maintained as extrachromosomal elements, or may be integrated into the 
host cell genome. Generation of stably transfected mammalian cell lines 
usually involves the latter; a DNA construct encoding a gene of interest 
along with a drug resistance gene (dominant selectable marker) is 
introduced into the host cell, and subsequent growth in the presence of 
the drug allows for the selection of cells that have successfully 
integrated the exogenous DNA. In many instances, the gene of interest is 
linked to a drug resistant selectable marker which can later be subjected 
to gene amplification. The gene encoding dihydrofolate reductase (DHFR) is 
most commonly used for this purpose. Growth of cells in the presence of 
methotrexate, a competitive inhibitor of DHFR, leads to increased DHFR 
production by means of amplification of the DHFR gene. As flanking regions 
of DNA will also become amplified, the resultant coamplification of a DHFR 
linked gene in the transfected cell line can lead to increased protein 
production, thereby resulting in high level expression of the gene of 
interest. 
While this approach has proven successful, there are a number of problems 
with the system because of the random nature of the integration event. 
These problems exist because expression levels are greatly influenced by 
the effects of the local genetic environment at the gene locus, a 
phenomena well documented in the literature and generally referred to as 
"position effects" (for example, see Al-Shawi et al, Mol. Cell. Biol., 
10:1192-1198 (1990); Yoshimura et al, Mol. Cell. Biol., 7:1296-1299 
(1987)). As the vast majority of mammalian DNA is in a transcriptionally 
inactive state, random integration methods offer no control over the 
transcriptional fate of the integrated DNA. Consequently, wide variations 
in the expression level of integrated genes can occur, depending on the 
site of integration. For example, integration of exogenous DNA into 
inactive, or transcriptionally "silent" regions of the genome will result 
in little or no expression. By contrast integration into a 
transcriptionally active site may result in high expression. 
Therefore, when the goal of the work is to obtain a high level of gene 
expression, as is typically the desired outcome of genetic engineering 
methods, it is generally necessary to screen large numbers of 
transfectants to find such a high producing clone. Additionally, random 
integration of exogenous DNA into the genome can in some instances disrupt 
important cellular genes, resulting in an altered phenotype. These factors 
can make the generation of high expressing stable mammalian cell lines a 
complicated and laborious process. 
Recently, our laboratory has described the use of DNA vectors containing 
translationally impaired dominant selectable markers in mammalian gene 
expression. (This is disclosed in U.S. Pat. No. 5,648,267. 
These vectors contain a translationally impaired neomycin 
phosphotransferase (neo) gene as the dominant selectable marker, 
artificially engineered to contain an intron into which a DHFR gene along 
with a gene or genes of interest is inserted. Use of these vectors as 
expression constructs has been found to significantly reduce the total 
number of drug resistant colonies produced, thereby facilitating the 
screening procedure in relation to conventional mammalian expression 
vectors. Furthermore, a significant percentage of the clones obtained 
using this system are high expressing clones. These results are apparently 
attributable to the modifications made to the neo selectable marker. Due 
to the translational impairment of the neo gene, transfected cells will 
not produce enough neo protein to survive drug selection, thereby 
decreasing the overall number of drug resistant colonies. Additionally, a 
higher percentage of the surviving clones will contain the expression 
vector integrated into sites in the genome where basal transcription 
levels are high, resulting in overproduction of neo, thereby allowing the 
cells to overcome the impairment of the neo gene. Concomitantly, the genes 
of interest linked to neo will be subject to similar elevated levels of 
transcription. This same advantage is also true as a result of the 
artificial intron created within neo; survival is dependent on the 
synthesis of a functional neo gene, which is in turn dependent on correct 
and efficient splicing of the neo introns. Moreover, these criteria are 
more likely to be met if the vector DNA has integrated into a region which 
is already highly transcriptionally active. 
Following integration of the vector into a transcriptionally active region, 
gene amplification is performed by selection for the DHFR gene. Using this 
system, it has been possible to obtain clones selected using low levels of 
methotrexate (50 nM), containing few (&lt;10) copies of the vector which 
secrete high levels of protein (&gt;55pg/cell/day). Furthermore, this can be 
achieved in a relatively short period of time. However, the success in 
amplification is variable. Some transcriptionally active sites cannot be 
amplified and therefore the frequency and extent of amplification from a 
particular site is not predictable. 
Overall, the use of these translationally impaired vectors represents a 
significant improvement over other methods of random integration. However, 
as discussed, the problem of lack of control over the integration site 
remains a significant concern. 
One approach to overcome the problems of random integration is by means of 
gene targeting, whereby the exogenous DNA is directed to a specific locus 
within the host genome. The exogenous DNA is inserted by means of 
homologous recombination occurring between sequences of DNA in the 
expression vector and the corresponding homologous sequence in the genome. 
However, while this type of recombination occurs at a high frequency 
naturally in yeast and other fungal organisms, in higher eukaryotic 
organisms it is an extremely rare event. In mammalian cells, the frequency 
of homologous versus non-homologous (random integration) recombination is 
reported to range from 1/100 to 1/5000 (for example, see Capecchi, 
Science, 244:1288-1292 (1989); Morrow and Kucherlapati, Curr. Op. 
Biotech., 4:577-582 (1993)). 
One of the earliest reports describing homologous recombination in 
mammalian cells comprised an artificial system created in mouse 
fibroblasts (Thomas et al, Cell, 44:419-428 (1986)). A cell line 
containing a mutated, non-functional version of the neo gene integrated 
into the host genome was created, and subsequently targeted with a second 
non-functional copy of neo containing a different mutation. Reconstruction 
of a functional neo gene could occur only by gene targeting. Homologous 
recombinants were identified by selecting for G418 resistant cells, and 
confirmed by analysis of genomic DNA isolated from the resistant clones. 
Recently, the use of homologous recombination to replace the heavy and 
light immunoglobulin genes at endogenous loci in antibody secreting cells 
has been reported. (U.S. Pat. No. 5,202,238, Fell et al, (1993).) However, 
this particular approach is not widely applicable, because it is limited 
to the production of immunoglobulins in cells which endogenously express 
immunoglobulins, e.g., B cells and myeloma cells. Also, expression is 
limited to single copy gene levels because co-amplification after 
homologous recombination is not included. The method is further 
complicated by the fact that two separate integration events are required 
to produce a functional immunoglobulin: one for the light chain gene 
followed by one for the heavy chain gene. 
An additional example of this type of system has been reported in NS/0 
cells, where recombinant immunoglobulins are expressed by homologous 
recombination into the immunoglobulin gamma 2A locus (Hollis et al, 
international patent application # PCT/IB95 (00014).) Expression levels 
obtained from this site were extremely high--on the order of 20pg/cell/day 
from a single copy integrant. However, as in the above example, expression 
is limited to this level because an amplifiable gene is not contegrated in 
this system. Also, other researchers have reported aberrant glycosylation 
of recombinant proteins expressed in NS/0 cells (for example, see Flesher 
et al, Biotech. and Bioeng., 48:399-407 (1995)), thereby limiting the 
applicability of this approach. 
The cre-loxP recombination system from bacteriophage P1 has recently been 
adapted and used as a means of gene targeting in eukaryotic cells. 
Specifically, the site specific integration of exogenous DNA into the 
Chinese hamster ovary (CHO) cell genome using cre recombinase and a series 
of lox containing vectors have been described. (Fukushige and Sauer, Proc. 
Natl. Acad. Sci. USA, 89:7905-7909 (1992).) This system is attractive in 
that it provides for reproducible expression at the same chromosomal 
location. However, no effort was made to identify a chromosomal site from 
which gene expression is optimal, and as in the above example, expression 
is limited to single copy levels in this system. Also, it is complicated 
by the fact that one needs to provide for expression of a functional 
recombinase enzyme in the mammalian cell. 
The use of homologous recombination between an introduced DNA sequence and 
its endogenous chromosomal locus has also been reported to provide a 
useful means of genetic manipulation in mammalian cells, as well as in 
yeast cells. (See e.g., Bradley et al, Meth. Enzymol., 223:855-879 (1993); 
Capecchi, Science, 244:1288-1292 (1989); Rothstein et al, Meth. Enzymol., 
194:281-301 (1991)). To date, most mammalian gene targeting studies have 
been directed toward gene disruption ("knockout") or site-specific 
mutagenesis of selected target gene loci in mouse embryonic stem (ES) 
cells. The creation of these "knockout" mouse models has enabled 
scientists to examine specific structure-function issues and examine the 
biological importance of a myriad of mouse genes. This field of research 
also has important implications in terms of potential gene therapy 
applications. 
Also, vectors have recently been reported by Cell-tech (Kent, U.K.) which 
purportedly are targeted to transcriptionally active sites in NSO cells, 
which do not require gene amplification (Peakman et al, Hum. Antibod. 
Hybridomas, 5:65-74 (1994)). However, levels of immunoglobulin secretion 
in these unamplified cells have not been reported to exceed 20pg/cell/day, 
while in amplified CHO cells, levels as high as 100pg/cell/day can be 
obtained (Id.). 
It would be highly desirable to develop a gene targeting system which 
reproducibly provided for the integration of exogenous DNA into a 
predetermined site in the genome known to be transcriptionally active. 
Also, it would be desirable if such a gene targeting system would further 
facilitate co-amplification of the inserted DNA after integration. The 
design of such a system would allow for the reproducible and high level 
expression of any cloned gene of interest in a mammalian cell, and 
undoubtedly would be of significant interest to many researchers. 
In this application, we provide a novel mammalian expression system, based 
on homologous recombination occurring between two artificial substrates 
contained in two different vectors. Specifically, this system uses a 
combination of two novel mammalian expression vectors, referred to as a 
"marking" vector and a "targeting" vector. 
Essentially, the marking vector enables the identification and marking of a 
site in the mammalian genome which is transcriptionally active, i.e., a 
site at which gene expression levels are high. This site can be regarded 
as a "hot spot" in the genome. After integration of the marking vector, 
the subject expression system enables another DNA to be integrated at this 
site, i.e., the targeting vector, by means of homologous recombination 
occurring between DNA sequences common to both vectors. This system 
affords significant advantages over other homologous recombination 
systems. 
Unlike most other homologous systems employed in mammalian cells, this 
system exhibits no background. Therefore, cells which have only undergone 
random integration of the vector do not survive the selection. Thus, any 
gene of interest cloned into the targeting plasmid is expressed at high 
levels from the marked hot spot. Accordingly, the subject method of gene 
expression substantially or completely eliminates the problems inherent to 
systems of random integration, discussed in detail above. Moreover, this 
system provides reproducible and high level expression of any recombinant 
protein at the same transcriptionally active site in the mammalian genome. 
In addition, gene amplification may be effected at this particular 
transcriptionally active site by including an amplifiable dominant 
selectable marker (e.g. DHFR) as part of the marking vector. 
OBJECTS OF THE INVENTION 
Thus, it is an object of the invention to provide an improved method for 
targeting a desired DNA to a specific site in a mammalian cell. 
It is a more specific object of the invention to provide a novel method for 
targeting a desired DNA to a specific site in a mammalian cell via 
homologous recombination. 
It is another specific object of the invention to provide novel vectors for 
achieving site specific integration of a desired DNA in a mammalian cell. 
It is still another object of the invention to provide novel mammalian cell 
lines which contain a desired DNA integrated at a predetermined site which 
provides for high expression. 
It is a more specific object of the invention to provide a novel method for 
achieving site specific integration of a desired DNA in a Chinese hamster 
ovary (CHO) cell. 
It is another more specific object of the invention to provide a novel 
method for integrating immunoglobulin genes, or any other genes, in 
mammalian cells at predetermined chromosomal sites that provide for high 
expression. 
It is another specific object of the invention to provide novel vectors and 
vector combinations suitable for integrating immunoglobulin genes into 
mammalian cells at predetermined sites that provide for high expression. 
It is another object of the invention to provide mammalian cell lines which 
contain immunoglobulin genes integrated at predetermined sites that 
provide for high expression. 
It is an even more specific object of the invention to provide a novel 
method for integrating immunoglobulin genes into CHO cells that provide 
for high expression, as well as novel vectors and vector combinations that 
provide for such integration of immunoglobulin genes into CHO cells. 
In addition, it is a specific object of the invention to provide novel CHO 
cell lines which contain immunoglobulin genes integrated at predetermined 
sites that provide for high expression, and have been amplified by 
methotrexate selection to secrete even greater amounts of functional 
immunoglobulins.

DETAILED DESCRIPTION OF THE INVENTION 
The invention provides a novel method for integrating a desired exogenous 
DNA at a target site within the genome of a mammalian cell via homologous 
recombination. Also, the invention provides novel vectors for achieving 
the site specific integration of a DNA at a target site in the genome of a 
mammalian cell. 
More specifically, the subject cloning method provides for site specific 
integration of a desired DNA in a mammalian cell by transfection of such 
cell with a "marker plasmid" which contains a unique sequence that is 
foreign to the mammalian cell genome and which provides a substrate for 
homologous recombination, followed by transfection with a "target plasmid" 
containing a sequence which provides for homologous recombination with the 
unique sequence contained in the marker plasmid, and further comprising a 
desired DNA that is to be integrated into the mammalian cell. Typically, 
the integrated DNA will encode a protein of interest, such as an 
immunoglobulin or other secreted mammalian glycoprotein. 
The exemplified homologous recombination system uses the neomycin 
phosphotransferase gene as a dominant selectable marker. This particular 
marker was utilized based on the following previously published 
observations; 
(i) the demonstrated ability to target and restore function to a mutated 
version of the neo gene (cited earlier) and 
(ii) our development of translationally impaired expression vectors, in 
which the neo gene has been artificially created as two exons with a gene 
of interest inserted in the intervening intron; neo exons are correctly 
spliced and translated in vivo, producing a functional protein and thereby 
conferring G418 resistance on the resultant cell population. In this 
application, the neo gene is split into three exons. The third exon of neo 
is present on the "marker" plasmid and becomes integrated into the host 
cell genome upon integration of the marker plasmid into the mammalian 
cells. Exons 1 and 2 are present on the targeting plasmid, and are 
separated by an intervening intron into which at least one gene of 
interest is cloned. Homologous recombination of the targeting vector with 
the integrated marking vector results in correct splicing of all three 
exons of the neo gene and thereby expression of a functional neo protein 
(as determined by selection for G418 resistant colonies). Prior to 
designing the current expression system, we had experimentally tested the 
functionality of such a triply spliced neo construct in mammalian cells. 
The results of this control experiment indicated that all three neo exons 
were properly spliced and therefore suggested the feasibility of the 
subject invention. 
However, while the present invention is exemplified using the neo gene, and 
more specifically a triple split neo gene, the general methodology should 
be efficacious with other dominant selectable markers. 
As discussed in greater detail infra, the present invention affords 
numerous advantages to conventional gene expression methods, including 
both random integration and gene targeting methods. Specifically, the 
subject invention provides a method which reproducibly allows for 
site-specific integration of a desired DNA into a transcriptionally active 
domain of a mammalian cell. Moreover, because the subject method 
introduces an artificial region of "homology" which acts as a unique 
substrate for homologous recombination and the insertion of a desired DNA, 
the efficacy of subject invention does not require that the cell 
endogenously contain or express a specific DNA. Thus, the method is 
generically applicable to all mammalian cells, and can be used to express 
any type of recombinant protein. 
The use of a triply spliced selectable marker, e.g., the exemplified triply 
spliced neo construct, guarantees that all G418 resistant colonies 
produced will arise from a homologous recombination event (random 
integrants will not produce a functional neo gene and consequently will 
not survive G418 selection). Thus, the subject invention makes it easy to 
screen for the desired homologous event. Furthermore, the frequency of 
additional random integrations in a cell that has under-gone a homologous 
recombination event appears to be low. 
Based on the foregoing, it is apparent that a significant advantage of the 
invention is that it substantially reduces the number of colonies that 
need be screened to identify high producer clones, i.e., cell lines 
containing a desired DNA which secrete the corresponding protein at high 
levels. On average, clones containing integrated desired DNA may be 
identified by screening about 5 to 20 colonies (compared to several 
thousand which must be screened when using standard random integration 
techniques, or several hundred using the previously described intronic 
insertion vectors) Additionally, as the site of integration was 
preselected and comprises a transcriptionally active domain, all exogenous 
DNA expressed at this site should produce comparable, i.e. high levels of 
the protein of interest. 
Moreover, the subject invention is further advantageous in that it enables 
an amplifiable gene to be inserted on integration of the marking vector. 
Thus, when a desired gene is targeted to this site via homologous 
recombination, the subject invention allows for expression of the gene to 
be further enhanced by gene amplification. In this regard, it has been 
reported in from the literature that different genomic sites have 
different capacities for gene amplification (Meinkoth et al, Mol. Cell 
Biol., 7:1415-1424 (1987)). Therefore, this technique is further 
advantageous as it allows for the placement of a desired gene of interest 
at a specific site that is both transcriptionally active and easily 
amplified. Therefore, this should significantly reduce the amount of time 
required to isolate such high producers. 
Specifically, while conventional methods for the construction of high 
expressing mammalian cell lines can take 6 to 9 months, the present 
invention allows for such clones to be isolated on average after only 
about 3-6 months. This is due to the fact that conventionally isolated 
clones typically must be subjected to at least three rounds of drug 
resistant gene amplification in order to reach satisfactory levels of gene 
expression. As the homologously produced clones are generated from a 
preselected site which is a high expression site, fewer rounds of 
amplification should be required before reaching a satisfactory level of 
production. 
Still further, the subject invention enables the reproducible selection of 
high producer clones wherein the vector is integrated at low copy number, 
typically single copy. This is advantageous as it enhances the stability 
of the clones and avoids other potential adverse side-effects associated 
with high copy number. As described supra, the subject homologous 
recombination system uses the combination of a "marker plasmid" and a 
"targeting plasmid" which are described in more detail below. The "marker 
plasmid" which is used to mark and identify a transcriptionally hot spot 
will comprise at least the following sequences: 
(i) a region of DNA that is heterologous or unique to the genome of the 
mammalian cell, which functions as a source of homology, allows for 
homologous recombination (with a DNA contained in a second target 
plasmid). More specifically, the unique region of DNA (i) will generally 
comprise a bacterial, viral, yeast synthetic, or other DNA which is not 
normally present in the mammalian cell genome and which further does not 
comprise significant homology or sequence identity to DNA contained in the 
genome of the mammalian cell. Essentially, this sequence should be 
sufficiently different to mammalian DNA that it will not significantly 
recombine with the host cell genome via homologous recombination. The size 
of such unique DNA will generally be at least about 2 to 10 kilobases in 
size, or higher, more preferably at least about 10 kb, as several other 
investigators have noted an increased frequency of targeted recombination 
as the size of the homology region is increased (Capecchi, Science, 
244:1288-1292 (1989)). 
The upper size limit of the unique DNA which acts as a site for homologous 
recombination with a sequence in the second target vector is largely 
dictated by potential stability constraints (if DNA is too large it may 
not be easily integrated into a chromosome) and the difficulties in 
working with very large DNAs. 
(ii) a DNA including a fragment of a selectable marker DNA, typically an 
exon of a dominant selectable marker gene. The only essential feature of 
this DNA is that it not encode a functional selectable marker protein 
unless it is expressed in association with a sequence contained in the 
target plasmid. Typically, the target plasmid will comprise the remaining 
exons of the dominant selectable marker gene (those not comprised in 
"targeting" plasmid). Essentially, a functional selectable marker should 
only be produced if homologous recombination occurs (resulting in the 
association and expression of this marker DNA (i) sequence together with 
the portion(s) of the selectable marker DNA fragment which is (are) 
contained in the target plasmid). 
As noted, the current invention exemplifies the use of the neomycin 
phosphotransferase gene as the dominant selectable marker which is "split" 
in the two vectors. However, other selectable markers should also be 
suitable, e.g., the Salmonella histidinol dehydrogenase gene, hygromycin 
phosphotransferase gene, herpes simplex virus thymidine kinase gene, 
adenosine deaminase gene, glutamine synthetase gene and 
hypoxanthine-guanine phosphoribosyl transferase gene. 
(iii) a DNA which encodes a functional selectable marker protein, which 
selectable marker is different from the selectable marker DNA (ii). This 
selectable marker provides for the successful selection of mammalian cells 
wherein the marker plasmid is successfully integrated into the cellular 
DNA. More preferably, it is desirable that the marker plasmid comprise two 
such dominant selectable marker DNAs, situated at opposite ends of the 
vector. This is advantageous as it enables integrants to be selected using 
different selection agents and further enables cells which contain the 
entire vector to be selected. Additionally, one marker can be an 
amplifiable marker to facilitate gene amplification as discussed 
previously. Any of the dominant selectable marker listed in (ii) can be 
used as well as others generally known in the art. 
Moreover, the marker plasmid may optionally further comprise a rare 
endonuclease restriction site. This is potentially desirable as this may 
facilitate cleavage. If present, such rare restriction site should be 
situated close to the middle of the unique region that acts as a substrate 
for homologous recombination. Preferably such sequence will be at least 
about 12 nucleotides. The introduction of a double stranded break by 
similar methodology has been reported to enhance the frequency of 
homologous recombination. (Choulika et al, Mol. Cell. Biol., 15:1968-1973 
(1995)). However, the presence of such sequence is not essential. 
The "targeting plasmid" will comprise at least the following sequences: 
(1) the same unique region of DNA contained in the marker plasmid or one 
having sufficient homology or sequence identity therewith that said DNA is 
capable of combining via homologous recombination with the unique region 
(i) in the marker plasmid. Suitable types of DNAs are described supra in 
the description of the unique region of DNA (1) in the marker plasmid. 
(2) The remaining exons of the dominant selectable marker, one exon of 
which is included as (ii) in the marker plasmid listed above. The 
essential features of this DNA fragment is that it result in a functional 
(selectable) marker protein only if the target plasmid integrates via 
homologous recombination (wherein such recombination results in the 
association of this DNA with the other fragment of the selectable marker 
DNA contained in the marker plasmid) and further that it allow for 
insertion of a desired exogenous DNA. Typically, this DNA will comprise 
the remaining exons of the selectable marker DNA which are separated by an 
intron. For example, this DNA may comprise the first two exons of the neo 
gene and the marker plasmid may comprise the third exon (back third of 
neo). 
(3) The target plasmid will also comprise a desired DNA, e.g., one encoding 
a desired polypeptide, preferably inserted within the selectable marker 
DNA fragment contained in the plasmid. Typically, the DNA will be inserted 
in an intron which is comprised between the exons of the selectable marker 
DNA. This ensures that the desired DNA is also integrated if homologous 
recombination of the target plasmid and the marker plasmid occurs. This 
intron may be naturally occurring or it may be engineered into the 
dominant selectable marker DNA fragment. 
This DNA will encode any desired protein, preferably one having 
pharmaceutical or other desirable properties. Most typically the DNA will 
encode a mammalian protein, and in the current examples provided, an 
immunoglobulin or an immunoadhesin. However the invention is not in any 
way limited to the production of immunoglobulins. 
As discussed previously, the subject cloning method is suitable for any 
mammalian cell as it does not require for efficacy that any specific 
mammalian sequence or sequences be present. In general, such mammalian 
cells will comprise those typically used for protein expression, e.g., CHO 
cells, myeloma cells, COS cells, BHK cells, Sp2/0 cells, NIH 3T3 and HeLa 
cells. In the examples which follow, CHO cells were utilized. The 
advantages thereof include the availability of suitable growth medium, 
their ability to grow efficiently and to high density in culture, and 
their ability to express mammalian proteins such as immunoglobulins in 
biologically active form. 
Further, CHO cells were selected in large part because of previous usage of 
such cells by the inventors for the expression of immunoglobulins (using 
the translationally impaired dominant selectable marker containing vectors 
described previously). Thus, the present laboratory has considerable 
experience in using such cells for expression. However, based on the 
examples which follow, it is reasonable to expect similar results will be 
obtained with other mammalian cells. 
In general, transformation or transfection of mammalian cells according to 
the subject invention will be effected according to conventional methods. 
So that the invention may be better understood, the construction of 
exemplary vectors and their usage in producing integrants is described in 
the examples below. 
EXAMPLE 1 
Design and Preparation of Marker and Targeting Plasmid DNA Vectors 
The marker plasmid herein referred to as "Desmond" was assembled from the 
following DNA elements: 
(a) Murine dihydrofolate reductase gene (DHFR), incorporated into a 
transcription cassette, comprising the mouse beta globin promoter 5" to 
the DHFR start site, and bovine growth hormone poly adenylation signal 3" 
to the stop codon. The DHFR transcriptional cassette was isolated from 
TCAE6, an expression vector created previously in this laboratory (Newman 
et al, 1992, Biotechnology, 10:1455-1460). 
(b) E. coli .beta.-galactosidase gene--commercially available, obtained 
from Promega as pSV-b-galactosidase control vector, catalog # E1081. 
(c) Baculovirus DNA, commercially available, purchased from Clontech as 
pBAKPAK8, cat # 6145-1. 
(d) Cassette comprising promoter and enhancer elements from Cytomegalovirus 
and SV40 virus. The cassette was generated by PCR using a derivative of 
expression vector TCAE8 (Reff et al, Blood, 83:435-445 (1994)). The 
enhancer cassette was inserted within the baculovirus sequence, which was 
first modified by the insertion of a multiple cloning site. 
(e) E. coli GUS (glucuronidase) gene, commercially available, purchased 
from Clontech as pB101, cat. # 6017-1. 
(f) E. coli luciferase gene, commercially available, obtained from Promega 
as pGEM-Luc (catalog # E1541). 
(g) S. typhimurium histidinol dehydrogenase gene (HisD). This gene was 
originally a gift from (Donahue et el, Gene, 18:47-59 (1982)), and has 
subsequently been incorporated into a transcription cassette comprising 
the mouse beta globin major promoter 5' to the gene, and the SV40 
polyadenylation signal 3' to the gene. 
The DNA elements described in (a)-(g) were combined into a pBR derived 
plasmid backbone to produce a 7.7 kb contiguous stretch of DNA referred to 
in the attached figures as "homology". Homology in this sense refers to 
sequences of DNA which are not part of the mammalian genome and are used 
to promote homologous recombination between transfected plasmids sharing 
the same homology DNA sequences. 
(h) Neomycin phosphotransferase gene from TN5 (Davis and Smith, Ann. Rev. 
Micro., 32:469-518 (1978)). The complete neo gene was subcloned into 
pBluescript SK-(Stratagene catalog # 212205) to facilitate genetic 
manipulation. A synthetic linker was then inserted into a unique Pst1 site 
occurring across the codons for amino acid 51 and 52 of neo. This linker 
encoded the necessary DNA elements to create an artificial splice donor 
site, intervening intron and splice acceptor site within the neo gene, 
thus creating two separate exons, presently referred to as neo exon 1 and 
2. Neo exon 1 encodes the first 51 amino acids of neo, while exon 2 
encodes the remaining 203 amino acids plus the stop codon of the protein A 
Not1 cloning site was also created within the intron. 
Neo exon 2 was further subdivided to produce neo exons 2 and 3. This was 
achieved as follows: A set of PCR primers were designed to amplify a 
region of DNA encoding neo exon 1, intron and the first 111 2/3 amino 
acids of exon2. The 3' PCR primer resulted in the introduction of a new 5' 
splice site immediately after the second nucleotide of the codon for amino 
acid 111 in exon 2, therefore generating a new smaller exon 2. The DNA 
fragment now encoding the original exon 1, intron and new exon 2 was then 
subcloned and propagated in a pBR based vector. The remainder of the 
original exon 2 was used as a template for another round of PCR 
amplification, which generated "exon3". The 5' primer for this round of 
amplification introduced a new splice acceptor site at the 5' side of the 
newly created exon 3, i.e. before the final nucleotide of the codon for 
amino acid 111. The resultant 3 exons of neo encode the following 
information: exon 1--the first 51 amino acids of neo; exon 2--the next 111 
2/3 amino acids, and exon 3 the final 91 1/3 amino acids plus the 
translational stop codon of the neo gene. 
Neo exon 3 was incorporated along with the above mentioned DNA elements 
into the marking plasmid "Desmond". Neo exons 1 and 2 were incorporated 
into the targeting plasmid "Molly". The Not1 cloning site created within 
the intron between exons 1 and 2 was used in subsequent cloning steps to 
insert genes of interest into the targeting plasmid. 
FIG. 1 depicts the arrangement of these DNA elements in the marker plasmid 
"Desmond". FIG. 2 depicts the arrangement of these elements in the first 
targeting plasmid, "Molly". FIG. 3 illustrates the possible arrangement in 
the CHO genome, of the various DNA elements after targeting and 
integration of Molly DNA into Desmond marked CHO cells. 
Construction of the marking and targeting plasmids from the above listed 
DNA elements was carried out following conventional cloning techniques 
(see, e.g., Molecular Cloning, A Laboratory Manual, J. Sambrook et al, 
1987, Cold Spring Harbor Laboratory Press, and Current Protocols in 
Molecular Biology, F. M. Ausubel et al, eds., 1987, John Wiley and Sons). 
All plasmids were propagated and maintained in E. coli XLI blue 
(Stratagene, cat. # 200236). Large scale plasmid preparations were 
prepared using Promega WIZARD MAXIPREP DNA PURIFICATION SYSTEM, according 
to the manufacturer's directions. 
EXAMPLE 2 
Construction of a Marked CHO Cell Line 
1. Cell Culture and Transfection Procedures to Produced Marked CHO Cell 
Line 
Marker plasmid DNA was linearized by digestion overnight at 37.degree. C. 
with Bst1107I. Linearized vector was ethanol precipitated and resuspended 
in sterile TE to a concentration of 1 mg/ml. Linearized vector was 
introduced into DHFR-Chinese hamster ovary cells (CHO cells) DG44 cells 
(Urlaub et al, Som. Cell and Mol. Gen., 12:555-566 (1986)) by 
electroporation as follows. 
Exponentially growing cells were harvested by centrifugation, washed once 
in ice cold SBS (sucrose buffered solution, 272 mM sucrose, 7 mM sodium 
phosphate, pH 7.4, 1 mM magnesium chloride) then resuspended in SBS to a 
concentration of 10.sup.7 cells/ml. After a 15 minute incubation on ice, 
0.4 ml of the cell suspension was mixed with 40 .mu.g linearized DNA in a 
disposable electroporation cuvette. Cells were shocked using a BTX 
electrocell manipulator (San Diego, Calif.) set at 230 volts, 400 
microfaraday capacitance, 13 ohm resistance. Shocked cells were then mixed 
with 20 ml of prewarmed CHO growth media (CHO-S-SFMII, Gibco/BRL, catalog 
# 31033-012) and plated in 96 well tissue culture plates. Forty eight 
hours after electroporation, plates were fed with selection media (in the 
case of transfection with Desmond, selection media is CHO-S-SFMII without 
hypoxanthine or thymidine, supplemented with 2 mM Histidinol (Sigma 
catalog # H6647)). Plates were maintained in selection media for up to 30 
days, until colonies appeared. These colonies were then removed from the 
96 well plates. Positive clones were expanded to tissue culture flasks and 
finally to 120 ml spinner flasks, and were maintained in selection media 
at all times. 
EXAMPLE 3 
Characterization of Marked CHO Cell Lines 
(a) Southern Analysis 
Genomic DNA was isolated from all stably growing Desmond marked CHO cells. 
DNA was isolated using the Invitrogen Easy.RTM. DNA kit, according to the 
manufacturer's directions. Genomic DNA was then digested with HindIII 
overnight at 37.degree. C., and subjected to Southern analysis using a PCR 
generated digoxygenin labelled probe specific to the DHFR gene. 
Hybridizations and washes were carried out using Boehringer Mannheim's DIG 
easy hyb (catalog # 1603 558) and DIG Wash and Block Buffer Set (catalog # 
1585 762) according to the manufacturer's directions. DNA samples 
containing a single band hybridizing to the DHFR probe were assumed to be 
Desmond clones which had integrated a single copy of the plasmid. These 
clones were retained for further analysis. Out of a total of 45 HisD 
resistant clones identified in the experiment, only 5 were single copy 
integrants. FIG. 4 shows a Southern blot containing all 5 of these single 
copy Desmond clones. Clone names are provided in the figure legend. 
(b) Northern Analysis 
Total RNA was isolated from all single copy Desmond clones using TRIzol 
reagent (Gibco/BRL cat # 15596-026) according to the manufacturer's 
directions. 10-20 .mu.g RNA from each clone was analyzed on duplicate 
formaldehyde gels. The resulting blots were probed with PCR generated 
digoxygenin labelled DNA probes to (i) DHFR message, (ii) HisD message and 
(iii) CAD message. CAD is a trifunctional protein involved in uridine 
biosynthesis (Wahl et al, J. Biol. Chem., 254, 17:8679-8689 (1979)), and 
is expressed equally in all cell types. It is used here as an internal 
control to help quantitate RNA loading. Hybridizations and washes were 
carried out using the above mentioned Boehringer Mannheim reagents. The 
results of the Northern analysis are shown in FIG. 5. The single copy 
Desmond clone exhibiting the highest levels of both the His D and DHFR 
message is clone 15C9, shown in lane 4 in both panels of the figure. This 
clone was designated as the "marked cell line" and used in future 
targeting experiments in CHO, examples of which are presented in the 
following sections. 
EXAMPLE 4 
Construction of a Marked SP2/0 Cell Line 
In order to demonstrate the utility of this expression system in any 
cultured mammalian cell, we have created a Desmond marked SP2/0 cell line. 
SP2/0 cells were grown in suspension culture in PFHM II media 
(protein-free hybridoma media, Gibco/BRL, cat # 12040-093). Exponentially 
growing cells were harvested by centrifugation, and all subsequent steps 
involved in preparation for electroporation, including preparation of 
Desmond plasmid DNA, were as described in the preceding section on marking 
CHO cells. Electroporation conditions were varied somewhat: ten 
electroporations were carried out at 400 volts, 25 .mu.Faradays 
capacitance and 13 ohms resistance, while an additional 10 were at 350 
volts, 50 .mu.Faradays and 13 ohms resistance. Each electroporation was 
then plated into individual 96 well plates. Selection media for these 
cells comprised PFHM II supplemented with 10 mM histidinol. Plates were 
maintained in selection media for 25 days. Resistant colonies were 
transferred from the 96 well dishes and ultimately expanded to spinner 
flasks. Experiments are currently ongoing to identify single copy SP2/0 
clones containing Desmond integrated in a transcriptionally active site. 
Future experiments will involve targeting such a site with Molly. 
EXAMPLE 5 
Expression of Anti-CD20 Antibody in Desmond Marked CHO Cells 
C2B8, a chimeric antibody which recognizes B-cell surface antigen CD20, has 
been cloned and expressed previously in our laboratory. (Reff et al, 
Blood, 83:434-45 (1994)). A 4.1 kb DNA fragment comprising the C2B8 light 
and heavy chain genes, along with the necessary regulatory elements 
(eukaryotic promoter and polyadenylation signals) was inserted into the 
artificial intron created between exons 1 and 2 of the neo gene contained 
in a pBR derived cloning vector. This newly generated 5 kb DNA fragment 
(comprising neo exon 1, C2B8 and neo exon 2) was excised and used to 
assemble the targeting plasmid Molly. The other DNA elements used in the 
construction of Molly are identical to those used to construct the marking 
plasmid Desmond, identified previously. A complete map of Molly is shown 
in FIG. 2. 
The targeting vector Molly was linearized prior to transfection by 
digestion with Kpn1 and Pac1, ethanol precipitated and resuspended in 
sterile TE to a concentration of 1.5 mg/mL. Linearized plasmid was 
introduced into exponentially growing Desmond marked cells essentially as 
described, except that 80 .mu.g DNA was used in each electroporation. 
Forty eight hours postelectroporation, 96 well plates were supplemented 
with selection medium--CHO-SSFMII supplemented with 400 .mu.g/mL Geneticin 
(G418, Gibco/BRL catalog # 10131-019). Plates were maintained in selection 
medium for up to 30 days, or until viable colonies were obtained. The 
supernatants from all G418 resistant colonies were assayed for C2B8 
production by standard ELISA techniques, and all productive clones were 
eventually expanded to 120 mL spinner flasks and further analyzed. 
Characterization of Antibody secreting Targeted Cells 
A total of 50 electroporations with Molly targeting plasmid were carried 
out in this experiment, each of which was plated into separate 96 well 
plates. A total of 10 viable, anti-CD20 antibody secreting clones were 
obtained and expanded to 120 ml spinner flasks. Genomic DNA was isolated 
from all clones, and Southern analyses were subsequently performed to 
determine whether the clones represented single homologous recombination 
events or whether additional random integrations had occurred in the same 
cells. The methods for DNA isolation and Southern hybridization were as 
described in the previous section. Genomic DNA was digested with EcoRI and 
probed with a PCR generated digoxygenin labelled probe to a segment of the 
CD20 heavy chain constant region. The results of this Southern analysis 
are presented in FIG. 6. As can be seen in the FIG. 8 of the 10 clones 
show a single band hybridizing to the CD20 probe, indicating a single 
homologous recombination event has occurred in these cells. Two of the 
ten, clones 24G2 and 28V9, show the presence of additional band(s), 
indicative of an additional random integration elsewhere in the genome. 
We examined the expression levels of anti-CD20 antibody in all ten of these 
clones, the data for which is shown in Table 1, below. 
TABLE 1 
______________________________________ 
Expression Level of Anti-CD20 Secreting Homologous Integrants 
Anti-CD20, 
Clone pg/c/d 
______________________________________ 
20F4 3.5 
21C7 1.3 
24G2 2.1 
25E1 2.4 
28C9 4.5 
29F9 0.8 
39G11 1.5 
42F9 1.8 
50G10 0.9 
5F9 0.3 
______________________________________ 
Expresion levels are reported as picogram per cell per day (pg/c/d) 
secreted by the individual clones, and represented the mean levels 
obtained from three separate ELISAs on samples taken from 120 mL spinner 
flasks. 
As can be seen from the data, there is a variation in antibody secretion of 
approximately ten fold between the highest and lowest clones. This was 
somewhat unexpected as we anticipated similar expression levels from all 
clones due to the fact the anti-CD20 genes are all integrated into the 
same Desmond marked site. Nevertheless, this observed range in expression 
extremely small in comparison to that seen using any traditional random 
integration method or with our translationally impaired vector system. We 
are currently investigating mRNA levels of all the clones in order to 
determine whether this expression range is due to differences in 
transcription levels. We anticipate that message levels will be similar in 
all clones and the observed differences in secretion levels among the 
homologous integrants is not an additional cellular factors which play a 
role in mediating secretion levels. This will of course be true in any 
expression system and is not inherent to this homologous system. 
Clone 20F4, the highest producing single copy integrant was selected for 
further study. Table 2 (below) presents ELISA and cell culture data from 
seven day production runs of this clone. 
TABLE 2 
______________________________________ 
7 Day Production Run Data for 20F4 
% Viable/ml 
T .times. 2 
Day Viable (.times. 10.sup.5) 
(hr) mg/L pg/c/d 
______________________________________ 
1 96 3.4 31 1.3 4.9 
2 94 6 29 2.5 3.4 
3 94 9.9 33 4.7 3.2 
4 90 17.4 30 6.8 3 
5 73 14 8.3 
6 17 3.5 9.5 
______________________________________ 
Clone 20F4 was seeded at 2 .times. 10.sup.5 ml in a 120 ml spinner flask 
on day 0. On the following six days, cell counts were taken, doubling 
times calculated and 1 ml samples of supernatant removed from the flask 
and analyzed for secreted antiCD20 by ELISA. 
This clone is secreting on average, 3-5 pg antibody/-cell/day, based on 
this ELISA data. This is the same level as obtained from other high 
expressing single copy clones obtained previously in our laboratory using 
the previously developed translationally impaired random integration 
vectors. This result indicates the following: 
(1) that the site in the CHO genome marked by the Desmond marking vector is 
highly transcriptionally active, and therefore represents an excellent 
site from which to express recombinant proteins, and 
(2) that targeting by means of homologous recombination can be accomplished 
using the subject vectors and occurs at a frequency high enough to make 
this system a viable and desirable alternative to random integration 
methods. 
To further demonstrate the efficacy of this system, we have also 
demonstrated that this site is amplifiable, resulting in even higher 
levels of gene expression and protein secretion. Amplification was 
achieved by plating serial dilutions of 20F4 cells, starting at a density 
of 2.5.times.10.sup.4 cells/ml, in 96 well tissue culture dishes, and 
culturing these cells in media (CHO-SSFMII) supplemented with 5, 10, 15 or 
20 nM methotrexate. Antibody secreting clones were screened using standard 
ELISA techniques, and the highest producing clones were expanded and 
further analyzed. A summary of this amplification experiment is presented 
in Table 3 below. 
TABLE 3 
______________________________________ 
Summary of 20F4 Amplification 
nM # Clones Expression Level 
# Clones 
Expression Level 
MTX Assayed mg/l 96 well 
Expanded 
pg/c/d from spinner 
______________________________________ 
10 56 3-13 4 10-15 
15 27 2-14 3 15-18 
20 17 4-11 1 ND 
______________________________________ 
Methotrexate amplification of 20F4 was set up as described in the text, 
using the concentrations of methotrexate indicated in the above table. 
Supernatants from all surviving 96 well colonies were assayed by ELISA, 
and the range of antiCD20 expressed by these clones is indicated in colum 
3. Based on these results, the highest producing clones were expanded to 
120 ml spinners and several ELISAs conducted on the spinner supernatants 
to determine the pg/cell/day expression levels, reported in column 5. 
The data here clearly demonstrates that this site can be amplified in the 
presence of methotrexate. Clones from the 10 and 15 nM amplifications were 
found to produce on the order of 15-20 pg/cell/day. It is anticipated that 
subsequent rounds of amplification on this clone will achieve higher 
levels, on the order of 60-100 pg/cell/-day, which is approaching the 
maximum secretion capacity of immunoglobulin in mammalian cells (Reff, M. 
E., Curr. Opin. Biotech., 4:573-576 (1993)). Further amplification of this 
clone is currently ongoing. 
Example 6 
Expression of Anti-Human CD23 Antibody in Desmond Marked CHO Cells 
CD23 is low affinity IgE receptor which mediates binding of IgE to B and T 
lymphocytes (Sutton, B. J., and Gould, H. J., Nature, 366:421-428 (1993)). 
Anti-human CD23 monoclonal antibody 5E8 is a human gamma-1 monoclonal 
antibody recently cloned and expressed in our laboratory. This antibody is 
disclosed in commonly assigned Ser. No. 08/803,085, filed on Feb. 20, 
1997. 
The heavy and light chain genes of 5E8 were cloned into the mammalian 
expression vector N5KG1, a derivative of the vector NEOSPLA (Barnett et 
al, in Antibody Expression and Engineering, H.Y Yang and T. Imanaka, eds., 
pp27-40 (1995)) and two modifications were then made to the genes. We have 
recently observed somewhat higher secretion of immunoglobulin light chains 
compared to heavy chains in other expression constructs in the laboratory 
(Reff et al, 1997, unpublished observations). In an attempt to compensate 
for this deficit, we altered the 5E8 heavy chain gene by the addition of a 
stronger promoter/enhancer element immediately upstream of the start site. 
Subsequently, a 2.9 kb DNA fragment comprising the 5E8 modified light and 
heavy chain genes was isolated from the N5KG1 vector and inserted between 
the BglII and SacII sites in the targeting vector Molly. Preparation of 
5E8-containing Molly and electroporation into Desmond 15C9 CHO cells was 
exactly as described in the preceding section. In this experiment, 20 
electroporations were performed and plated into 96 well tissue culture 
dishes. Three anti-CD23 antibody secreting homologous clones were isolated 
and expanded to 120 ml spinner flasks. The levels of antibody secretion 
obtained from these clones ranged from 2-4 pg/cell/day, in close agreement 
with the expression levels observed for anti-CD20. Methotrexate 
amplification of the highest anti-CD23 expressing clone is currently 
underway. 
EXAMPLE 7 
Expression of Immunoadhesin in Desmond Marked CHO Cells 
CTLA-4, a member of the Ig superfamily, is found on the surface of T 
lymphocytes and is thought to play a role in antigen-specific T-cell 
activation (Dariavach et al, Eur. J. Immunol., 18:1901-1905 (1988); and 
Linsley et al, J. Exp. Med., 174:561-569 (1991)). In order to further 
study the precise role of the CTLA-4 molecule in the activation pathway, a 
soluble fusion protein comprising the extracellular domain of CTLA-4 
linked to a truncated form of the human IgG1 constant region was created 
(Linsley et al (Id.). We have recently expressed this CTLA-4 Ig fusion 
protein in the mammalian expression vector BLECH1, a derivative of the 
plasmid NEOSPLA (Barnett et al, in Antibody Expression and Engineering, 
H.Y Yang and T. Imanaka, eds., pp27-40 (1995)). An 800 bp fragment 
encoding the CTLA-4 Ig was isolated from this vector and inserted between 
the SacII and BglII sites in Molly. 
Preparation of CTLA-4Ig-Molly and electroporation into Desmond clone 15C9 
CHO cells was performed as described in the preceding section. Twenty 
electroporations were carried out, and plated into 96 well culture dishes 
as described previously. Eighteen CTLA-4 expressing clones were isolated 
from the 96 well plates and carried forward to the 120 ml spinner stage. 
Southern analyses on genomic DNA isolated from each of these clones was 
then carried out to determine how many of the homologous clones contained 
additional random integrants. Genomic DNA was digested with BglII and 
probed with a PCR generated digoxygenin labelled probe to the human IgG1 
constant region. The results of this analysis indicated that 85% of the 
CTLA-4 clones are homologous integrants only; the remaining 15% contained 
one additional random integrant. This result corroborates the findings 
from the expression of anti-CD20 discussed above, where 80% of the clones 
were single homologous integrants. Therefore, we can conclude that this 
expression system reproducibly yields single targeted homologous 
integrants in at least 80% of all clones produced. 
Expression levels for the homologous CTlA4-Ig clones ranged from 8-12 
pg/cell/day. This is somewhat higher than the range reported for anti-CD20 
antibody and anti-CD23 antibody clones discussed above. However, we have 
previously observed that expression of this molecule using the intronic 
insertion vector system also resulted in significantly higher expression 
levels than are obtained for immunoglobulins. We are currently unable to 
provide an explanation for this observation. 
Example 8 
Targeting Anti-CD20 to an alternate Desmond Marked CHO Cell Line 
As we described in a preceding section, we obtained 5 single copy Desmond 
marked CHO cell lines (see FIGS. 4 and 5). In order to demonstrate that 
the success of our targeting strategy is not due to some unique property 
of Desmond clone 15C9 and limited only to this clone, we introduced 
anti-CD20 Molly into Desmond clone 9B2 (lane 6 in FIG. 4, lane 1 in FIG. 
5). Preparation of Molly DNA and electroporation into Desmond 9B2 was 
exactly as described in the previous sections. We obtained one homologous 
integrant from this experiment. This clone was expanded to a 120 ml 
spinner flask, where it produced on average 1.2 pg anti-CD20/cell/day. 
This is considerably lower expression than we observed with Molly targeted 
into Desmond 15C9. However, this was the anticipated result, based on our 
northern analysis of the Desmond clones. As can be seen in FIG. 5, mRNA 
levels from clone 9B2 are considerably lower than those from 15C9, 
indicating the site in this clone is not as transcriptionally active as 
that in 15C9. Therefore, this experiment not only demonstrates the 
reproducibility of the system--presumably any marked Desmond site can be 
targeted with Molly--it also confirms the northern data that the site in 
Desmond 15C9 is the most transcriptionally active. 
From the foregoing, it will be appreciated that, although specific 
embodiments of the invention have been described herein for purposes of 
illustration, various modifications may be made without diverting from the 
scope of the invention. Accordingly, the invention is not limited by the 
appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14683 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTTCTAGACCTAGGGCGGCCAGCTAGTAGCTTTGCTTCTCAATTTCTTATTTGCATAATG60 
AGAAAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGC120 
CAAAAAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAG180 
GGAGTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATA240 
TGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCT300 
CACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTT360 
GCTCCTCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCA420 
GGGCTGCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTA480 
TCCCCGCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGG540 
GGATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCC600 
AAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGA660 
AAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTC720 
TCAGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATG780 
ATGCCTTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAG840 
TCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTG900 
TGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGA960 
AATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCA1020 
TCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCT1080 
CTGCTCCCCTCCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGA1140 
TCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT1200 
TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCA1260 
TCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG1320 
GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCA1380 
GCTGGGGCTCGAAGCGGCCGCCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACG1440 
CGGCGGGGACCGTCACACTGAGGTTTTCCGCCAGACGCCACTGCTGCCAGGCGCTGATGT1500 
GCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAGCCAGAGTT1560 
GCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTTCAATCAACTGTTTACCTTGTGGAG1620 
CGACATCCAGAGGCACTTCACCGCTTGCTAGCGGCTTACCATCCAGCGCCACCATCCAGT1680 
GCAGGAGCTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCC1740 
CGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTTTTGCTTCCGTCAGCGCTGGATGCG1800 
GCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGAACTGGCGATCGTTCGGCGTATCAC1860 
CAAAATCACCGCCGTAAGCCGACCACGGGTTGCCGTTTTCATCATATTTAATCAGCGACT1920 
GATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACGAAACGCCTGCC1980 
AGTATTTAGCGAAACCGCCAAGACTGTTACCCATCGCGTGGGCGTATTCGCAAAGGATCA2040 
GCGGGCGCGTCTCTCCGGGTAGCGAAAGCCATTTTTTGATGGACCATTTCGGACCAGCCG2100 
GGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGG2160 
TGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGGAAGGATCGACAGATTTGATCCAGC2220 
GATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGACCAGATGATCA2280 
CACTCGGGTGATTACGATCGCGCTGCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTA2340 
GCCAGCGCGGATCATCGGTCAGACGATTCATTGGCACCATGCCGTGGGTTTCAATATTGG2400 
CTTCATCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCG2460 
GATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCTGCTTCATCAGCAGGATATCCTGCA2520 
CCATCGTCTGCTCATCCATGACCTGACCATGCAGAGGATGATGCTCGTGACGGTTAACGC2580 
CTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGCAGACCATTTCCAATCCGCACCTCGC2640 
GGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTGTGCAGTTCAA2700 
CCACCGCACGATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTTCA2760 
GACGCAGTGTGACGCGATCGGCATAACCACCAGGCTCATCGATAATTTCACCGCCGAAAG2820 
GCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGT2880 
AGTCACGCAACTCGCCGCACATCTGAACTTCAGCCTCCAGTACAGCGCGGCTGAAATCAT2940 
CATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTATGCAGCAACG3000 
AGACGTCACGGAAAATGCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGT3060 
CACTCCAACGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCA3120 
GGTCAAATTCAGACGGCAAACGACTGTCCTGGCTGTAACCGACCCACGCCCCGTTGCACC3180 
ACAGATGAAACGCCGAGTTAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTGTAGCC3240 
AGCTTTCATCAACATTAAATGTGAGCGAGTAACAACCCGTCGGATTCTCCGTGGGAACAA3300 
ACGGCGGATTGACCGTAATGGGATAGGTTACGTTGGTGTAGATGGGCGCATCGTAACCGT3360 
GCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCC3420 
AGCTTTCCGGCACTGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGC3480 
TGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGA3540 
AAGCGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGAC3600 
GTTGTAAAACGACTTAATCCGTCGAGGGGCTGCCTCGAAGCAGACGACCTTCCGTTGTGC3660 
AGCCAGCGGCGCCTGCGCCGGTGCCCACAATCGTGCGCGAACAAACTAAACCAGAACAAA3720 
TCATACCGGCGGCACCGCCGCCACCACCTTCTCCTGTGCCTAACATTCCAGCGCCTCCAC3780 
CACTACCACCACCATCGATGTCTGAATTGCCGCCCGCTCCACCAATGCCGACGGAACCTC3840 
AACCCGCTGCACCTTTAGACGACAGACAACAATTGTTGGAAGCTATTAGAAACGAAAAAA3900 
ATCGCACTCGTCTCAGACCGGCTCTCTTAAGGTAGCTCAAACCAAAAACGGCGCCCGAAA3960 
CCAGTACAATAGTTGAGGTGCCGACTGTGTTGCCTAAAGAGACATTTGAGCTTAAACCGC4020 
CGTCTGCACCACCGCCACCACCTCCGCCTCCGCCTCCGCCGCCAGCCCCGCCTGCGCCTC4080 
CACCGATGGTAGATTCATCATCAGCTCCACCACCGCCGCCATTAGTAGATTTGCCGTCTG4140 
AAATGTTACCACCGCCTGCACCATCGCTTTCTAACGTGTTGTCTGAATTAAAATCGGGCA4200 
CAGTTAGATTGAAACCCGCCCAAAAACGCCCGCAATCAGAAATAATTCCAAAAAGCTCAA4260 
CTACAAATTTGATCGCGGACGTGTTAGCCGACACAATTAATAGGCGTCGTGTGGCTATGG4320 
CAAAATCGTCTTCGGAAGCAACTTCTAACGACGAGGGTTGGGACGACGACGATAATCGGC4380 
CTAATAAAGCTAACACGCCCGATGTTAAATATGTCCAAGCTACTAGTGGTACCTTAATTA4440 
AGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGC4500 
GGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAG4560 
CCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTG4620 
CCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAAT4680 
TCCCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG4740 
TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCTCAACGACCCCCGC4800 
CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA4860 
CGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT4920 
ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC4980 
CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT5040 
ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCA5100 
CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGAAGCTTGGCC5160 
GGCCATATAAACGGCGGCCAGCTTTATTTAACGTGTTTACGTCGAGTCAATTGTACACTA5220 
ACGACAGTGATGAAAGAAATACAAAAGCGCATAATATTTTGAACGACGTCGAACCTTTAT5280 
TACAAAACAAAACACAAACGAATATCGACAAAGCTAGATTGCTGCTACAAGATTTGGCAA5340 
GTTTTGTGGCGTTGAGCGAAAATCCATTAGATAGTCCAGCCATCGGTTCGGAAAAACAAC5400 
CCTTGTTTGAAACTAATCGAAACCTATTTTACAAATCTATTGAGGATTTAATATTTAAAT5460 
TCAGATATAAAGACGCTGAAAATCATTTGATTTTCGCTCTAACATACCACCCTAAAGATT5520 
ATAAATTTAATGAATTATTAAAATACATCAGCAACTATATATTGATAGACATTTCCAGTT5580 
TGTGATATTAGTTTGTGCGTCTCATTACAATGGCTGTTATTTTTAACAACAAACAACTGC5640 
TCGCAGACAATAGTATAGAAAAGGGAGGTGAACTGTTTTTGTTTAACGGTTCGTACAACA5700 
TTTTGGAAAGTTATGTTAATCCGGTGCTGCTAAAAAATGGTGTAATTGAACTAGAAGAAG5760 
CTGCGTACTATGCCGGCAACATATTGTACAAAACCGACGATCCCAAATTCATTGATTATA5820 
TAAATTTAATAATTAAAGCAACACACTCCGAAGAACTACCAGAAAATAGCACTGTTGTAA5880 
ATTACAGAAAAACTATGCGCAGCGGTACTATACACCCCATTAAAAAAGACATATATATTT5940 
ATGACAACAAAAAATTTACTCTATACGATAGATACATATATGGATACGATAATAACTATG6000 
TTAATTTTTATGAGGAGAAAAATGAAAAAGAGAAGGAATACGAAGAAGAAGACGACAAGG6060 
CGTCTAGTTTATGTGAAAATAAAATTATATTGTCGCAAATTAACTGTGAATCATTTGAAA6120 
ATGATTTTAAATATTACCTCAGCGATTATAACTACGCGTTTTCAATTATAGATAACACTA6180 
CAAATGTTCTTGTTGCGTTTGGTTTGTATCGTTAATAAAAAACAAATTTAGCATTTATAA6240 
TTGTTTTATTATTCAATAATTACAAATAGGATTGAGACCCTTGCAGTTGCCAGCAAACGG6300 
ACAGAGCTTGTCGAGGAGAGTTGTTGATTCATTGTTTGCCTCCCTGCTGCGGTTTTTGAC6360 
CGAAGTTCATGCCAGTCCAGCGTTTTTGCAGCAGAAAAGCCGCCGACTTCGGTTTGCGGT6420 
CGCGAGTGAAGATCCCTTTCTTGTTACCGCCAACGCGCAATATGCCTTGCGAGGTCGCAA6480 
AATCGGCGAAATTCCATACCTGTTCACCGACGACGGCGCTGACGCGATCAAAGACGCGGT6540 
GATACATATCCAGCCATGCACACTGATACTCTTCACTCCACATGTCGGTGTACATTGAGT6600 
GCAGCCCGGCTAACGTATCCACGCCGTATTCGGTGATGATAATCGGCTGATGCAGTTTCT6660 
CCTGCCAGGCCAGAAGTTCTTTTTCCAGTACCTTCTCTGCCGTTTCCAAATCGCCGCTTT6720 
GGACATACCATCCGTAATAACGGTTCAGGCACAGCACATCAAAGAGATCGCTGATGGTAT6780 
CGGTGTGAGCGTCGCAGAACATTACATTGACGCAGGTGATCGGACGCGTCGGGTCGAGTT6840 
TACGCGTTGCTTCCGCCAGTGGCGCGAAATATTCCCGTGCACCTTGCGGACGGGTATCCG6900 
GTTCGTTGGCAATACTCCACATCACCACGCTTGGGTGGTTTTTGTCACGCGCTATCAGCT6960 
CTTTAATCGCCTGTAAGTGCGCTTGGTGAGTTTCCCCGTTGACTGCCTCTTCGTTGTACA7020 
GTTCTTTCGGCTTGTTGCCCGCTTCGAAACCAATGCCTAAAGAGAGGTTAAAGCCGACAG7080 
CAGCAGTTTCATCAATCACCACGATGCCATGTTCATCTGCCCAGTCGAGCATCTCTTCAG7140 
CGTAAGGGTAATGCGAGGTACGGTAGGAGTTGGCCCTAATCCAGTCCATTAATGCGTGGT7200 
CGTGCACCATCAGCACGTTATCGAATCCTTTGCCACGCAAGTCCGCATCTTCATGACGAC7260 
CAAAGCCAGTAAAGTAGAACGGTTTGTGGTTAATCAGGAACTGTTCGCCCTTCACTGCCA7320 
CTGACCGGATGCCGACGCGAAGCGGGTAGATATCACACTCTGTCTGGCTTTTGGCTGTGA7380 
CGCACAGTTCATAGAGATAACCTTCACCCGGTTGCCAGAGGTGCGGATTCACCACTTGCA7440 
AAGTCCCGCTAGTGCCTTGTCCAGTTGCAACCACCTGTTGATCCGCATCACGCAGTTCAA7500 
CGCTGACATCACCATTGGCCACCACCTGCCAGTCAACAGACGCGTGGTTACAGTCTTGCG7560 
CGACATGCGTCACTACGGTGATATCGTCCACCCAGGTGTTCGGCGTGGTGTAGAGCATTA7620 
CGCTGCGATGGATTCCGGCATAGTTAAAGAAATCATGGAAGTAAGATTGCTTTTTCTTGC7680 
CGTTTTCGTTGGTAATCACCATTCCCGGCGGGATAGTCTGCCAGTTCAGTTCGTTGTTCA7740 
CACAAACGGTGATACCCCTCGACGGATTAAAGACTTCAAGCGGTCAACTATGAAGAAGTG7800 
TTCGTCTTCGTCCCAGTAAGCTATGTCTCTAGAATGTAGCCATCCATCCTTGTCAATCAA7860 
GGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGTCCTCTGACACA7920 
TAATACGCCTCTCTGATTAACGCCCAGCGTTTTCCCGGTATCCAGATCCACAACCTTCGC7980 
TTCAAAAAATGGAACAACTTTACCGACCGCGCCCGGTTTATCATCCCCCTCGGGTGTAAT8040 
CAGAATAGCTGATGTAGTCTCAGTGAGCCCATATCCTTGTCGTATCCCTGGAAGATGGAA8100 
GCGTTTTGCAACCGCTTCCCCGACTTCTTTCGAAAGAGGTGCGCCCCCAGAAGCAATTTC8160 
GTGTAAATTAGATAAATCGTATTTGTCAATCAGAGTGCTTTTGGCGAAGAATGAAAATAG8220 
GGTTGGTACTAGCAACGCACTTTGAATTTTGTAATCCTGAAGGGATCGTAAAAACAGCTC8280 
TTCTTCAAATCTATACATTAAGACGACTCGAAATCTACATATCAAATATCCGAGTGTAGT8340 
AAACATTCCAAAACCGTGATGGAATGGAACAACACTTAAAATCGCAGTATCCGGAATGAT8400 
TTGATTGCCAAAAATAGGATCTCTGGCATGCGAGAATCTAGCGCAGGCAGTTCTATGCGG8460 
AAGGGCCACACCCTTAGGTAACCCAGTAGATCCAGAGGAATTGTTTTGTCACGATCAAAG8520 
GACTCTGGTACAAAATCGTATTCATTAAAACCGGGAGGTAGATGAGATGTGACGAAGGTG8580 
TACATCGACTGAAATCCCTGGTAATCCGTTTTAGAATCCATGATAATAATTTTCTGGATT8640 
ATTGGTAATTTTTTTTGCACGTTCAAAATTTTTTGCAACCCCTTTTTGGAAACAAACACT8700 
ACGGTAGGCTGCGAAATGTTCATACTGTTGAGCAATTCACGTTCATTATAAATGTCGTTC8760 
GCGGGCGCAACTGCAACTCCGATAAATAACGCGCCCAACACCGGCATAAAGAATTGAAGA8820 
GAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGCCCATATCGTTTCATAGCT8880 
TCTGCCAACCGAACGGACATTTCGAAGTATTCCGCGTACGTGATGTTCACCTCGATATGT8940 
GCATCTGTAAAAGGAATTGTTCCAGGAACCAGGGCGTATCTCTTCATAGCCTTATGCAGT9000 
TGCTCTCCAGCGGTTCCATTCTCTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGA9060 
AAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAA9120 
AAAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATCATTCAGAGGGA9180 
GTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGC9240 
TTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCAC9300 
ACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCT9360 
CCTCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATCGATCCACCATGGGCTTC9420 
AATACCCTGATTGACTGGAACAGCTGTAGCCCTGAACAGCAGCGTGCGCTGCTGACGCGT9480 
CCGGCGATTTCCGCCTCTGACAGTATTACCCGGACGGTCAGCGATATTCTGGATAATGCA9540 
AAAACGCGCGGTGACGATGCCCTGCGTGAATACAGCGCTAAATTTGATAAAACAGAAGTG9600 
ACAGCGCTACGCGTCACCCCTGAAGAGATCGCCGCCGCCGGCGCGCGTCTGAGCGACGAA9660 
TTAAAACAGGCGATGACCGCTGCCGTCAAAAATATTGAAACGTTCCATTCCGCGCAGACG9720 
CTACCGCTTGTAGATGTGGAAACCCAGCCAGGCGTGCGTTGCCAGCAGGTTACGCGTCCC9780 
GTCTCGTCTGTCGGTCTGTATATTCCCGGCGGCTCGGCTCCGCTCTTCTCAACGGTGCTG9840 
ATGCTGGCGACGCCGGCGCGCATTGCGGGATGCTAGAAGGTGGTTCTGTGCTCGCCGCCG9900 
CCCATCGCTGATGAAATCCTCTATGCGGCGCAACTGTGTGGCGTGCAGGAATTCTTTAAC9960 
CTCGGCGGCGCGCAGGCGATTGCCGCTCTGGCCTTCGGCAGCGAGTCCGTACCGAAAGTG10020 
GATAAAATTTTTGGCCCCGGCAACGCCTTTGTAACCGAAGCCAAACGTCAGGTCAGCCAG10080 
CGTCTCGACGGCGCGGCTATCGATATGCCAGCCGAGCCGTCTGAAGTACTGGTGATCGCA10140 
GACAGCGGCGCAACACCGGATTTCGTCGCTTCTGACCTGCTCTCCCAGACTGAGCACGGC10200 
CCGGATTCCCAGGTGATCCTGCTGACGCCTGATGCTGACATTGCCCGCAAGGTGGCGGAG10260 
GCGGTAGAACGTCAACTGGCGGAACTGCCGCGCGCGGACACCGCCTGGCAGGCCCTGAGC10320 
GCCAGTCGTCTGATTGTGACCAAAGATTTAGCGCAGTGCGTCGCCATCTCTAATCAGTAT10380 
GGGCCGGAACACTTAATCATCCAGACGCGCAATGCGCGCGATTTGGTGGATGCGATTACC10440 
AGCGCAGGCTCGGTATTTCTCGGCGACTGGTCGCCGGAATCCGCCGGTGATTACGCTTCC10500 
GGAACCAACCATGTTTTACCGACCTATGGCCATACTGCTACCTGTTCCAGCCTTGGGTTA10560 
GCGGATTTCCAGAAACGGATGACCGTTCAGGAACTGTCGAAAGCGGGCTTTTCCGCTCTG10620 
GCATCAACCATTGAAACATTGGCGGGGGCAGAACGTCTGACCGCCCATAAAAATGCCGTG10680 
ACCCTGCGCGTAAACGCCCTCAAGGAGCAAGCATGAGCACTGAAAACACTCTCAGCGTCG10740 
CTGACTTAGCCCGTGAAAATGTCCGCAACCTGGAGATCCAGACATGATAAGATACATTGA10800 
TGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTG10860 
TGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAA10920 
TTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTA10980 
AAACCTCTACAAATGTGGTATGGCTGATTATGATCTCTAGCTCGACGGGGCGCCTGGCCG11040 
CTACTAACTCTCTCCTCCCTCCTTTTTCCTGCAGGCTCAAGGCGCGCATGCCCGACGGCG11100 
AGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC11160 
GCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAG11220 
CGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCG11280 
TGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACG11340 
AGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCC11400 
ATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTT11460 
CCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCA11520 
CCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT11580 
CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATCT11640 
ATCTTATCATGTCTGGATCGCGGCCGGTCTCTCTCTAGCCCTAGGTCTAGACTTGGCAGA11700 
ACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTG11760 
GGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGC11820 
GGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACT11880 
GCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTT11940 
CGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCG12000 
CAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATT12060 
GACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCAC12120 
AACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTC12180 
GTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGA12240 
CCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGC12300 
TTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTC12360 
ACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAA12420 
ACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGA12480 
GCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGA12540 
CCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGAT12600 
TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATA12660 
CCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT12720 
GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGA12780 
TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC12840 
CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG12900 
CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG12960 
AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT13020 
TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGT13080 
GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG13140 
CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT13200 
GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT13260 
CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCT13320 
GCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC13380 
CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATC13440 
TCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG13500 
TTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA13560 
AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCA13620 
ATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC13680 
CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGC13740 
TGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC13800 
AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT13860 
TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGT13920 
TGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTC13980 
CGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAG14040 
CTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT14100 
TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC14160 
TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG14220 
CCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT14280 
TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC14340 
GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC14400 
TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA14460 
ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTG14520 
TCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG14580 
CACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAAC14640 
CTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAA14683 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18986 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TTAATTAAGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGT60 
TAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGC120 
TGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCAT180 
ACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAG240 
AATTAATTCCCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGGTCATAGCCCATA300 
TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA360 
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTT420 
CCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT480 
GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA540 
TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT600 
CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTT660 
TGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGAAG720 
CTTGGCCGGCCATATAAACGGCGGCCAGCTTTATTTAACGTGTTTACGTCGAGTCAATTG780 
TACACTAACGACAGTGATGAAAGAAATACAAAAGCGCATAATATTTTGAACGACGTCGAA840 
CCTTTATTACAAAACAAAACACAAACGAATATCGACAAAGCTAGATTGCTGCTACAAGAT900 
TTGGCAAGTTTTGTGGCGTTGAGCGAAAATCCATTAGATAGTCCAGCCATCGGTTCGGAA960 
AAACAACCCTTGTTTGAAACTAATCGAAACCTATTTTACAAATCTATTGAGGATTTAATA1020 
TTTAAATTCAGATATAAAGACGCTGAAAATCATTTGATTTTCGCTCTAACATACCACCCT1080 
AAAGATTATAAATTTAATGAATTATTAAAATACATCAGCAACTATATATTGATAGACATT1140 
TCCAGTTTGTGATATTAGTTTGTGCGTCTCATTACAATGGCTGTTATTTTTAACAACAAA1200 
CAACTGCTCGCAGACAATAGTATAGAAAAGGGAGGTGAACTGTTTTTGTTTAACGGTTCG1260 
TACAACATTTTGGAAAGTTATGTTAATCCGGTGCTGCTAAAAAATGGTGTAATTGAACTA1320 
GAAGAAGCTGCGTACTATGCCGGCAACATATTGTACAAAACCGACGATCCCAAATTCATT1380 
GATTATATAAATTTAATAATTAAAGCAACACACTCCGAAGAACTACCAGAAAATAGCACT1440 
GTTGTAAATTACAGAAAAACTATGCGCAGCGGTACTATACACCCCATTAAAAAAGACATA1500 
TATATTTATGACAACAAAAAATTTACTCTATACGATAGATACATATATGGATACGATAAT1560 
AACTATGTTAATTTTTATGAGGAGAAAAATGAAAAAGAGAAGGAATACGAAGAAGAAGAC1620 
GACAAGGCGTCTAGTTTATGTGAAAATAAAATTATATTGTCGCAAATTAACTGTGAATCA1680 
TTTGAAAATGATTTTAAATATTACCTCAGCGATTATAACTACGCGTTTTCAATTATAGAT1740 
AATACTACAAATGTTCTTGTTGCGTTTGGTTTGTATCGTTAATAAAAAACAAATTTAGCA1800 
TTTATAATTGTTTTATTATTCAATAATTACAAATAGGATTGAGACCCTTGCAGTTGCCAG1860 
CAAACGGACAGAGCTTGTCGAGGAGAGTTGTTGATTCATTGTTTGCCTCCCTGCTGCGGT1920 
TTTTCACCGAAGTTCATGCCAGTCCAGCGTTTTTGCAGCAGAAAAGCCGCCGACTTCGGT1980 
TTGCGGTCGCGAGTGAAGATCCCTTTCTTGTTACCGCCAACGCGCAATATGCCTTGCGAG2040 
GTCGCAAAATCGGCGAAATTCCATACCTGTTCACCGACGACGGCGCTGACGCGATCAAAG2100 
ACGCGGTGATACATATCCAGCCATGCACACTGATACTCTTCACTCCACATGTCGGTGTAC2160 
ATTGAGTGCAGCCCGGCTAACGTATCCACGCCGTATTCGGTGATGATAATCGGCTGATGC2220 
AGTTTCTCCTGCCAGGCCAGAAGTTCTTTTTCCAGTACCTTCTCTGCCGTTTCCAAATCG2280 
CCGCTTTGGACATACCATCCGTAATAACGGTTCAGGCACAGCACATCAAAGAGATCGCTG2340 
ATGGTATCGGTGTGAGCGTCGCAGAACATTACATTGACGCAGGTGATCGGACGCGTCGGG2400 
TCGAGTTTACGCGTTGCTTCCGCCAGTGGCGCGAAATATTCCCGTGCACCTTGCGGACGG2460 
GTATCCGGTTCGTTGGCAATACTCCACATCACCACGCTTGGGTGGTTTTTGTCACGCGCT2520 
ATCAGCTCTTTAATCGCCTGTAAGTGCGCTTGCTGAGTTTCCCCGTTGACTGCCTCTTCG2580 
CTGTACAGTTCTTTCGGCTTGTTGCCCGCTTCGAAACCAATGCCTAAAGAGAGGTTAAAG2640 
CCGACAGCAGCAGTTTCATCAATCACCACGATGCCATGTTCATCTGCCCAGTCGAGCATC2700 
TCTTCAGCGTAAGGGTAATGCGAGGTACGGTAGGAGTTGGCCCCAATCCAGTCCATTAAT2760 
GCGTGGTCGTGCACCATCAGCACGTTATCGAATCCTTTGCCACGCAAGTCCGCATCTTCA2820 
TGACGACCAAAGCCAGTAAAGTAGAACGGTTTGTGGTTAATCAGGAACTGTTCGCCCTTC2880 
ACTGCCACTGACCGGATGCCGACGCGAAGCGGGTAGATATCACACTCTGTCTGGCTTTTG2940 
GCTGTGACGCACAGTTCATAGAGATAACCTTCACCCGGTTGCCAGAGGTGCGGATTCACC3000 
ACTTGCAAAGTCCCGCTAGTGCCTTGTCCAGTTGCAACCACCTGTTGATCCGCATCACGC3060 
AGTTCAACGCTGACATCACCATTGGCCACCACCTGCCAGTCAACAGACGCGTGGTTACAG3120 
TCTTGCGCGACATGCGTCACCACGGTGATATCGTCCACCCAGGTGTTCGGCGTGGTGTAG3180 
AGCATTACGCTGCGATGGATTCCGGCATAGTTAAAGAAATCATGGAAGTAAGACTGCTTT3240 
TTCTTGCCGTTTTCGTCGGTAATCACCATTCCCGGCGGGATAGTCTGCCAGTTCAGTTCG3300 
TTGTTCACACAAACGGTGATACCCCTCGACGGATTAAAGACTTCAAGCGGTCAACTATGA3360 
AGAAGTGTTCGTCTTCGTCCCAGTAAGCTATGTCTCCAGAATGTAGCCATCCATCCTTGT3420 
CAATCAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGTCCTC3480 
TGACACATAATTCGCCTCTCTGATTAACGCCCAGCGTTTTCCCGGTATCCAGATCCACAA3540 
CCTTCGCTTCAAAAAATGGAACAACTTTACCGACCGCGCCCGGTTTATCATCCCCCTCGG3600 
GTGTAATCAGAATAGCTGATGTAGTCTCAGTGAGCCCATATCCTTGTCGTATCCCTGGAA3660 
GATGGAAGCGTTTTGCAACCGCTTCCCCGACTTCTTTCGAAAGAGGTGCGCCCCCAGAAG3720 
CAATTTCGTGTAAATTAGATAAATCGTATTTGTCAATCAGAGTGCTTTTGGCGAAGAATG3780 
AAAATAGGGTTGGTACTAGCAACGCACTTTGAATTTTGTAATCCTGAAGGGATCGTAAAA3840 
ACAGCTCTTCTTCAAATCTATACATTAAGACGACTCGAAATCCACATATCAAATATCCGA3900 
GTGTAGTAAACATTCCAAAACCGTGATGGAATGGAACAACACTTAAAATCGCAGTATCCG3960 
GAATGATTTGATTGCCAAAAATAGGATCTCTGGCATGCGAGAATCTAGCGCAGGCAGTTC4020 
TATGCGGAAGGGCCACACCCTTAGGTAACCCAGTAGATCCAGAGGAATTGTTTTGTCACG4080 
ATCAAAGGACTCTGGTACAAAATCGTATTCATTAAAACCGGGAGGTAGATGAGATGTGAC4140 
GAACGTGTACATCGACTGAAATCCCTGGTAATCCGTTTTAGAATCCATGATAATAATTTT4200 
CTGGATTATTGGTAATTTTTTTTGCACGTTCAAAATTTTTTGCAACCCCTTTTTGGAAAC4260 
AAACACTACGGTAGGCTGCGAAATGTTCATACTGTTGAGCAATTCACGTTCATTATAAAT4320 
GTCGTTCGCGGGCGCAACTGCAACTCCGATAAATAACGCGCCCAACACCGGCATAAAGAA4380 
TTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGCCCATATCGTTT4440 
CATAGCTTCTGCCAACCGAACGGACATTTCGAAGTATTCCGCGTACGTGATGTTCACCTC4500 
GATATGTGCATCTGTAAAAGGAATTGTTCCAGGAACCAGGGCGTATCTCTTCATAGCCTT4560 
ATGCAGTTGCTCTCCAGCGGTTCCATCCTCTAGCTTTGCTTCTCAATTTCTTATTTGCAT4620 
AATGAGAAAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCG4680 
TTGCCAAAAAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTC4740 
AGAGGGAGTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGA4800 
AATATGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATC4860 
TGCTCACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATC4920 
AGTTGCTCCTCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATCGATCCACCAT4980 
GGGCTTCAATACCCTGATTGACTGGAACAGCTGTAGCCCTGAACAGCAGCGTGCGCTGCT5040 
GACGCGTCCGGCGATTTCCGCCTCTGACAGTATTACCCGGACGGTCAGCGATATTCTGGA5100 
TAATGTAAAAACGCGCGGTGACGATGCCCTGCGTGAATACAGCGCTAAATTTGATAAAAC5160 
AGAAGTGACAGCGCTACGCGTCACCCCTGAAGAGATCGCCGCCGCCGGCGCGCGTCTGAG5220 
CGACGAATTAAAACAGGCGATGACCGCTGCCGTCAAAAATATTGAAACGTTCCATTCCGC5280 
GCAGACGCTACCGCCTGTAGATGTGGAAACCCAGCCAGGCGTGCGTTGCCAGCAGGTTAC5340 
GCGTCCCGTCTCGTCTGTCGGTCTGTATATTCCCGGCGGCTCGGCTCCGCTCTTCTCAAC5400 
GGTGCTGATGCTGGCGACGCCGGCGCGCATTGCGGGATGCCAGAAGGTGGTTCTGTGCTC5460 
GCCGCCGCCCATCGCTGATGAAATCCTCTATGCGGCGCAACTGTGTGGCGTGCAGGAAAT5520 
CTTTAACGTCGGCGGCGCGCAGGCGATTGCCGCTCTGGCCTTCGGCAGCGAGTCCGTACC5580 
GAAAGTGGATAAAATTTTTGGCCCCGGCAACGCCTTTGTAACCGAAGCCAAACGTCAGGT5640 
CAGCCAGCGTCTCGACGGCGCGGCTATCGATATGCCAGCCGGGCCGTCTGAAGTACTGGT5700 
GATCGCAGACAGCGGCGCAACACCGGATTTCGTCGCTTCTGACCTGCTCTCCCAGGCTGA5760 
GCACGGCCCGGATTCCCAGGTGATCCTGCTGACGCCTGATGCTGACATTGCCCGCAAGGT5820 
GGCGGAGGCGGTAGAACGTCAACTGGCGGAACTGCCGCGCGCGGACACCGCCCGGCAGGC5880 
CCTGAGCGCCAGTCGTCTGATTGTGACCAAAGATTTAGCGCAGTGCGTCGCCATCTCTAA5940 
TCAGTATGGGCCGGAACACTTAATCATCCAGACGCGCAATGCGCGCGATTTGGTGGATGC6000 
GATTACCAGCGCAGGCTCGGTATTTCTCGGCGACTGGTCGCCGGAATCCGCCGGTGATTA6060 
CGCTTCCGGAACCAACCATGTTTTACCGACCTATGGCTATACTGCTACCTGTTCCAGCCT6120 
TGGGTTAGCGGATTTCCAGAAACGGATGACCGTTCAGGAACTGTCGAAAGCGGGCTTTTC6180 
CGCTCTGGCATCAACCATTGAAACATTGGCGGCGGCAGAACGTCTGACCGCCCATAAAAA6240 
TGCCGTGACCCTGCGCGTAAACGCCCTCAAGGAGCAAGCATGAGCACTGAAAACACTCTC6300 
AGCGTCGCTGACTTAGCCCGTGAAAATGTCCGCAACCTGGAGATCCAGACATGATAAGAT6360 
ACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTG6420 
AAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACA6480 
ACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAA6540 
GCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCTCTAGCTCGACGGCGCGC6600 
CTCTAGAGCAGTGTGGTTTTGCAAGAGGAAGCAAAAAGCCTCTCCACCCAGGCCTGGAAT6660 
GTTTCCACCCAATGTCGAGCAGTGTGGTTTTGCAAGAGGAAGCAAAAAGCCTCTCCACCC6720 
AGGCCTGGAATGTTTCCACCCAATGTCGAGCAAACCCCGCCCAGCGTCTTGTCATTGGCG6780 
AATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGTCCCACTTCGCATATTAAG6840 
GTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCCAATATGGGATCGGCCATTGA6900 
ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGA6960 
CTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG7020 
GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGTAAG7080 
TGCGGCCGTCGATGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGC7140 
CATGCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTT7200 
AGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCT7260 
GGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATA7320 
CTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGA7380 
ATTAATTCCCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT7440 
ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC7500 
CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC7560 
CATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTG7620 
TATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT7680 
TATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAGCTATTAGTC7740 
ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT7800 
GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC7860 
CAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC7920 
GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTGAACCGTCAGATC7980 
GCCTGGAGACGCCATCACAGATCTCTCACTATGGATTTTCAGGTGCAGATTATCAGCTTC8040 
CTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGACAAATTGTTCTCTCCCAGTCTCCA8100 
GCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGT8160 
GTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTAT8220 
GCCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACT8280 
TCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAG8340 
CAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACG8400 
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT8460 
GCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG8520 
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG8580 
GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC8640 
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC8700 
AACAGGGGAGAGTGTTGAATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTC8760 
GTGACAACATGCGGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGC8820 
CAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC8880 
ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT8940 
ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG9000 
CATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCTATGCCAAGTA9060 
CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA9120 
CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG9180 
TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC9240 
CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT9300 
TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT9360 
GGGAGGTCTATATAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACAC9420 
AGACCCGTCGACATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGT9480 
GTCCTGTCCCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCA9540 
GTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTA9600 
AAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGAT9660 
ACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGC9720 
ACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCA9780 
AGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTC9840 
ACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG9900 
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG9960 
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC10020 
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG10080 
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAG10140 
AAAGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA10200 
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC10260 
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC10320 
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG10380 
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG10440 
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG10500 
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA10560 
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT10620 
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC10680 
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC10740 
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC10800 
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCGTTAACGGT10860 
TACCAACTACCTAGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGTATGATCAG10920 
CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT10980 
TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGC11040 
ATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGG11100 
AGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTG11160 
GGGCTCGACAGCAACGCTAGGTCGAGGCCGCTACTAACTCTCTCCTCCCTCCTTTTTCCT11220 
GCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGT11280 
GCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCA11340 
GGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAAT11400 
GCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCG11460 
CATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGA11520 
AGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGTAAGTGAGCTCCAATTCAAG11580 
CTTCCTAGGGCGGCCAGCTAGTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAA11640 
AAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAA11700 
AGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGT11760 
ACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTT11820 
GTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACAC11880 
AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC11940 
TCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCT12000 
GCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCC12060 
GCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATT12120 
GGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGA12180 
ATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACC12240 
TGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGT12300 
AGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCC12360 
TTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGA12420 
GGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACA12480 
AGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATAT12540 
AAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAG12600 
TATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCT12660 
CCCCTCCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGC12720 
CTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTT12780 
GACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA12840 
TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA12900 
GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGG12960 
GGCTCGAAGCGGCCGCCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCG13020 
GGGAGCGTCACACTGAGGTTTTCCGCCAGACGCCACTGCTGCCAGGCGCTGATGTGCCCG13080 
GCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAGCCAGAGTTGCCCG13140 
GCGCTCTCCGGCTGCGGTAGTTCAGGCAGTTCAATCAACTGTTTACCTTGTGGACCGACA13200 
TCCAGAGGCACTTCACCGCTTGCCAGCGGCTTACCATCCAGCGCCACCATCCAGTGCAGG13260 
AGCTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCCCGGAT13320 
AAACGGAACTGGAAAAACTGCTGCTGGTGTTTTGCTTCCGTCAGCGCTGGATGCGGCGTG13380 
CGGTCGGCAAAGACCAGACCGTTCATACAGAACTGGCGATCGTTCGGCGTATCGCCAAAA13440 
TCACCGCCGTAAGCCGACCACGGGTTGCCGTTTTCATCATATTTAATCAGCGACTGATCC13500 
ACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACGAAACGCCTGCCAGTAT13560 
TTAGCGAAACCGCCAAGACTGTTACCCATCGCTGGGGCGTATTCGCAAAGGATCAGCGGG13620 
CGCGTCTCTCCGGGTAGCGAAAGCCATTTTTTGATGGACCATTTCGGACCAGCCGGGAAG13680 
GGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGGTGTCG13740 
GCTCCGCCGCCTTCATACTGCACCGGGCGGGAAGGATCGACAGATTTGATCCAGCGATAC13800 
AGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGACCAGATGATCACACTC13860 
GGGTGATTACGATCGCGCTGCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTAGCCAG13920 
CGCGGATCATCGGTCAGACGATTCATTGGCACCATGCCGTGGGTTTCAATATTGGCTTCA13980 
TCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCGGATAA14040 
TGCCAACAGCGCACGGCGTTAAAGTTGTTCTGCTTCATCAGCAGGATATCCTGCACCATC14100 
GTCTGCTCATCCATGACCTGACCATGCAGAGGATGATGCTCGTGACGGTTAACGCCTCGA14160 
ATCAGCAACGGCTTGCCGTTCAGCAGCAGCAGACCATTTTCAATCCGCACCTCGCGGAAA14220 
CCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTGTGCAGTTCAACCACC14280 
GCACGATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTTCAGACGC14340 
AGTGTGACGCGATCGGCATAACCACCACGCTCATCGATAATTTCACCGCCGAAAGGCGCG14400 
GTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCA14460 
CGCAACTCGCCGCACATCTGAACTTCAGCCTCCAGTACAGCGCGGCTGAAATCATCATTA14520 
AAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTATGCAGCAACGAGACG14580 
TCACGGAAAATGCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTC14640 
CAACGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCAGGTCA14700 
AATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCACGCCCCGTTGCACCACAGA14760 
TGAAACGCCGAGTTAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTGTAGCCAGCTT14820 
TCATCAACATTAAATGTGAGCGAGTAACAACCCGTCGGATTCTCCGTGGGAACAAACGGC14880 
GGATTGACCGTAATGGGATAGGTTACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATC14940 
TGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCCAGCTT15000 
TCCGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGC15060 
AACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG15120 
GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT15180 
AAAACGACTTAATCCGTCGAGGGGCTGCCTCGAAGCAGACGACCTTCCGTTGTGCAGCCA15240 
GCGGCGCCTGCGCCGGTGCCCACAATCGTGCGCGAACAAACTAAACCAGAACAAATTATA15300 
CCGGCGGCACCGCCGCCACCACCTTCTCCCGTGCCTAACATTCCAGCGCCTCCACCACCA15360 
CCACCACCATCGATGTCTGAATTGCCGCCCGCTCCACCAATGCCGACGGAACCTCAACCC15420 
GCTGCACCTTTAGACGACAGACAACAATTGTTGGAAGCTATTAGAAACGAAAAAAATCGC15480 
ACTCGTCTCAGACCGGTCAAACCAAAAACGGCGCCCGAAACCAGTACAATAGTTGAGGTG15540 
CCGACTGTGTTGCCTAAAGAGACATTTGAGCCTAAACCGCCGTCTGCATCACCGCCACCA15600 
CCTCCGCCTCCGCCTCCGCCGCCAGCCCCGCCTGCGCCTCCACCGATGGTAGATTTATCA15660 
TCAGCTCCACCACCGCCGCCATTAGTAGATTTGCCGTCTGAAATGTTACCACCGCCTGCA15720 
CCATCGCTTTCTAACGTGTTGTCTGAATTAAAATCGGGCACAGTTAGATTGAAACCCGCC15780 
CAAAAACGCCCGCAATCAGAAATAATTCCAAAAAGCTCAACTACAAATTTGATCGCGGAC15840 
GTGTTAGCCGACACAATTAATAGGCGTCGTGTGGCTATGGCAAAATCGTCTTCGGAAGCA15900 
ACTTCTAACGACGAGGGTTGGGACGACGACGATAATCGGCCTAATAAAGCTAACACGCCC15960 
GATGTTAAATATGTCCAAGCTACTAGTGGTACCGCTTGGCAGAACATATCCATCGCGTCC16020 
GCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTG16080 
CGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGT16140 
TAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCT16200 
GCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACG16260 
CGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTA16320 
CCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTT16380 
CTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGG16440 
GCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATT16500 
ACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAACC16560 
GCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAAC16620 
GAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAG16680 
CTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAG16740 
CTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAG16800 
GGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGAT16860 
AGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACC16920 
ATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTT16980 
CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAG17040 
CTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA17100 
TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT17160 
TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC17220 
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT17280 
CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG17340 
TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA17400 
AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT17460 
ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA17520 
ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA17580 
ACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT17640 
TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT17700 
TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA17760 
TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA17820 
TGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT17880 
CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG17940 
CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT18000 
AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG18060 
ACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC18120 
GCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG18180 
CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCA18240 
TCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA18300 
GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA18360 
TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA18420 
ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA18480 
AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGG18540 
ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG18600 
GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG18660 
CACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAG18720 
GAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC18780 
TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA18840 
TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG18900 
TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTA18960 
TCACGAGGCCCTTTCGTCTTCAAGAA18986 
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