Patent Publication Number: US-2003232358-A1

Title: Method of identifying genes controlling differentiation

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     [0001] This application claims priority from U.S. provisional patent application S. No. 60/365,359 filed Mar. 15, 2002. 
    
    
     
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002] Not applicable.  
       BACKGROUND OF THE INVENTION  
       [0003] Modem cell biology includes a variety of techniques to manipulate various cells of living organisms in vitro. A particularly significant and intriguing category of cell culture are known as stem cells. Stem cells are undifferentiated, or only partially differentiated cells, that have the capability to differentiate into a number of progenitor cell types. The term stem cells can be used to refer to a cell type which is the progenitor of a category of a cell type in a larger organism, such as a hematopoietic stem cell, or can refer to a totally undifferentiated stem cell which, at least in theory, has the ability to differentiate into any of the tissues of the body of the whole organism. Stem cell cultures have been developed from a variety of tissues and in a number of different animals.  
       [0004] Recently it has become possible to generate, culture, and maintain cultures of primate embryonic stem cells, including human embryonic stem cells. For example, see U.S. Pat. Nos. 5,843,780 and 6,200,806 to Thomson. Primate embryonic stem cells are stem cell cultures, originally created from cells taken from embryos, that survive indefinitely in culture and are made up of cells which have the capability of differentiating into the major tissue types of a primate body. Primate embryonic stem cells can be maintained in an undifferentiated state in culture, or can be allowed to begin a differentiation process by which the cells become committed to one or another developmental cell lineage. Typically the differentiation of stem cells into different tissue types begins with the creation of embryoid bodies, which causes the stem cells in the embryoid body to begin to differentiate into different cell types in different portions of the embryoid body. In fact, maintaining human embryonic stem cells in an undifferentiated state requires careful attention to culture conditions since the cells will spontaneously begin uncontrolled differentiation if the culture conditions are incorrect.  
       [0005] One of the significant areas of research enabled by the development of stem cells is to begin to try to understand what genes or factors cause undifferentiated cells to begin to differentiate into committed cell lineages. It is theorized that for most initial differentiation of stem cells that a single genetic factor turns on, or off, causing the stem cell thereafter to begin to express some genes, and not others, and to thereby acquire a commitment toward a particular cell lineage. Identifying the genetic factors responsible for this initial differentiation step is a non-trivial problem. Yet scientifically, this inquiry is important in understanding the initial development of living organisms.  
       [0006] It is possible to do comparative RNA analysis between stem cells and cells which have made the first step in differentiation, and identify what species of RNA are produced in the progeny cell that are not produced in the stem cell. A comparative RNA expression study makes it is possible to know what genes are turned on when a cell commits to a specific lineage as compared to the undifferentiated stem cell from which it arose. However, having a catalog of the genes which turn on does not help to distinguish the gene or genes which initiated the process from the larger number of gene which are turned on as a result of the process. In fact, it may be quite difficult or impractical using comparative RNA analysis to identify a gene which initiates the differentiation process since intracellular factors, such as transcription factors, need not be produced in great abundance to have the effect of causing a change in cell differentiation. Proven methods have not been available heretofore to identify the factors which are responsible for primary cell differentiation.  
       BRIEF SUMMARY OF THE INVENTION  
       [0007] The present invention is summarized in that a method is described for identifying the cellular factors responsible for cell differentiation from a beginning cell type to a target cell type. The method begins with the random cloning of expressed genes by use of a cDNA library, the cDNA being from the target cell type. The cDNA genes are transferred into expression vectors effective in cells of the beginning cell type. The expression vectors are transferred into the beginning cells and then the cells are cultured in a way so as to permit differentiation into the target cell type. Those cells which have differentiated to the desired target cell type are identified, preferably through the use of a selectable marker. Then the DNA is recovered from the differentiated cells and that DNA is analyzed to determine what inserted cDNA caused the differentiation to the target cell type. In this way, the cellular factors responsible for a specific single cell differentiation can be identified.  
       [0008] It is an object of the present invention to provide the a method to identify the factors responsible for primary cell differentiation.  
       [0009] It is a feature of the present invention that it permits the identification of the genetic factors responsible for the initial stages of cellular differentiation beginning from human embryonic stem cells.  
       [0010] Other objects, advantages, and features of the present invention will become apparent from the following specification. 
     
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
     [0011]FIGS. 1 and 2 are illustrations of vectors adapted for use in the method of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0012] The present invention is intended to identify the genes or genetic factors which are responsible for the primary differentiation of undifferentiated cells to differentiated or partially differentiated cells. The method described here may also be used to identify the genes or genetic factors which cause initially differentiated cells to differentiate further into various cell lineages in the body. The method, which is referred to here as expression cloning, makes use of a gene expression library which is placed in expression vectors and inserted into the undifferentiated cells of interest. Then the undifferentiated cells are permitted to differentiate. Those cells which have differentiated into the cell type of specific interest are then identified. Once the targeted cells have been identified, conventional DNA characterization techniques can be used to identify which cDNA species causes the differentiated cells to differentiate into the cell type of interest.  
     [0013] The present invention was developed to permit the identification of the genetic factors responsible for the first stages of cell differentiation, i.e. to identify those factors which cause the most undifferentiated cells, pluripotent stem cells, to begin the process of differentiation into the various tissues of the body. In particular, using human stem cells, it is possible to use this process to determine what genetic factors cause the stem cells to begin the differentiation process into the various cell lineages which ultimately make up all the cells of the human body. While the techniques and details of the method were developed to permit the application of the process to human embryonic stem cells, it is contemplated that the process can be used at many levels of differentiation with many cells types in culture, from a beginning undifferentiated cell to a terminally differentiated target cell type. The term human undifferentiated stem cell is used here to refer to cells which have the developmental potential of human embryonic stem cells, specifically including cells are derived from other sources such as human embryonic germline cells and stem cells derived from mature or adult bodies.  
     [0014] The process of the present invention thus begins with selecting a beginning cell type, such as an undifferentiated cell type, and a target cell type, such as a cell which has undergone one differentiation step from a stem cell to the precursor of some other type of cell. For purposes of having an example, assume that the target cell type is a cell which has become committed to neural cell lineage, but which is otherwise undifferentiated, a cell type which will be referred to here as a neural precursor cell.  
     [0015] The next step in the process is to select a library of expressed genes from the cells. There are two broad ways to accomplish this objective, to make a library or to use a reference library. Typically, when collections of nucleic acids indicative of gene expression is desired, one uses or makes a cDNA library for the tissue, cell or organism in question. A cDNA library is typically made from the mRNA species which are present in cells in the lineage of the target cell type and is most suitably made from the target cell type itself. The other alternative is to use a reference library collection. For example, a collaborative scientific effort is underway, under the guidance of the U.S. National Institutes of Health, to establish a gene collection to be known as the Mammalian Gene Collection (MGC). The MGC would be a defined gene expression library, intended to overcome some of the limitations in the use of mRNA libraries made for an individual experiment or investigation. The MGC will include clones, identifiers and sequences for the full-length transcripts from mouse and human cDNA libraries. The use of a clone set from a reference library ensures that the clones will be full length, and avoids two common problems with laboratory created mRNA libraries. The problems are that an mRNA library will tend to over-represent the abundant genes expressed in the cell from which the library is made and that the clones in the library will often not be full-length. In general, because of the more fair representation of cDNA species, and the full-length of the members of clones, it is expected that the use of a reference library of expressed genes will generally be more efficient and preferred. The use of the reference library also permits the identification of subsets of clones or genes to preferentially examine desired categories of genes, such as transcription regulators, in preference to other genes or to limit the number of clones which must be created. So, again in the neural precursor cell example, the cDNA library is made by either method to represent the mRNA species present in terminally differentiated nerve cells, neural precursor cells, or any cells located in the lineage between the two.  
     [0016] While this method may be used to detect genes responsible for differentiation, it may be used in a reverse sense as well. If the beginning cell is a differentiated cell, and the target cell is a undifferentiated stem cell, the method can be used to identify genes controlling status of a cell as a stem cell.  
     [0017] Then the cDNA library species are cloned into expression vectors capable of expression in primate undifferentiated stem cells. As it turns out, many mammalian gene expression vectors do not work well in stem cells. Thus the selection of the expression vector is a critical parameter, and will be discussed in more detail below.  
     [0018] The expression vector not only includes the cDNA species to be expressed in the transfected stem cells, it also includes a marker gene system that can be used to detect successful transformants. Such a marker system is needed, as will be appreciated from the discussion below, to identify the cells from the stem cell culture which have undergone the desired differentiation step. Marker gene systems, which can include screenable markers which permit cells to be screened for transformants, or selectable markers which permit a selection agent to select for transformants, permit the identification of transformant cells which express the marker gene. For a screenable marker, such as the green fluorescent protein gene which confers fluorescence on expressing cells, the culture of cells is screened for expression of a detectable phenotype, such as cell fluorescence. For a selectable marker, such as a gene for antibiotic resistance, the culture of cells is exposed to an selection agent, such as an antibiotic, which is toxic to all cells except those expressing the selectable marker gene, in this case one for antibiotic resistance. The use of such marker systems is a common practice in gene transformation processes and many other types and example of markers are known in the art.  
     [0019] It is also preferred that the marker system be under the control of a tissue specific promoter specific to the cell type of the target cell. If, for example, a selectable antibiotic resistance gene is used in a process to identify genes responsible for differentiation to neural precursor cells, the antibiotic resistance gene would be under the control of a tissue specific promoter which only expresses the gene it controls in nerve cells. In this way, the marker will be expressed only when the cell into which it is transformed has differentiated into the target cell type.  
     [0020] Thus the process proceeds as follows. The cDNA library is created and cloned into the expression vector system. The expression vectors, including both the cDNA library and the marker system, are transformed into cells of the cell type. The beginning cell type culture is then cultured. It is preferred that this culture not include other conditions which favor cell differentiation. In fact, it is preferred that the cell culture at this step favors cells remaining undifferentiated. In that way, only cells which are caused to differentiate by the presence of the inserted and expressing cDNA will actually differentiate. The differentiated cells can be detected in a number of ways. One way is simply to examine the cells for morphological change consistent with the desired differentiation step. The preferred way is to use the marker system, which was included in the expression vector for just this purpose. The marker system is used to detect which cells are then expressing the marker system, indicating that the tissue specific promoter driving the marker system has commenced to drive expression. This indicates that the cells have differentiated into the target cells. At this point it should be true that the cDNA species which was transformed into this particular cell or cells was responsible for the differentiation of the beginning cell type into the target cells type. It is now necessary to identify what the cDNA was.  
     [0021] This next step is performed most easily by a PCR reaction. The expression vector has previously been characterized so the 5′ and 3′ flanking regions in the vector around the cDNA segment are known. So DNA is recovered from the differentiated cell or cells and a PCR process in performed on the recovered DNA using primers selected from the flanking regions in the expression vector which lie on either side of the cDNA insert. The product of the PCR process will be amplified DNA extending from one primer to the other and thus extending across the cDNA insert. By sequencing the DNA of the PCR reaction product, the DNA sequence of the cDNA insert that caused the cell differentiation can be determined. Assuming that the cell was transformed only by one expression vector, this will indicate that this single cDNA encodes a protein which, when expressed in an undifferentiated cell, causes the differentiation of that cell toward the target cell type. In other words, this process permits the identification of single genetic factors responsible for single steps of cell lineage differentiation.  
     [0022] Note that this method thus required screening a number of clones to find the clones that were associated with differentiation events. Since screening large numbers of clones can be burdensome, note again that the concept of using a well-defined library makes the overall process more efficient. In a laboratory-made library, the number of full-length clones can vary. Since the screening here is for relatively rare events, the more the library is limited to only include the genes likely to be interest, the shorter the search is likely to be for the gene of interest. With a non-random or defined library, it is possible to start with a library where the number of members in the library is a manageable number. For example, the human genome is thought currently to have only about 50,000 open reading frames. If the clones in the library is even more restricted, to cover only species likely to be involved in control of transcription, for example, the number can be further reduced.  
     [0023] As stated earlier, this expression cloning technique requires the use of a cloning vector which works in the undifferentiated cell type. With regard to the study of human embryonic stem cells, finding an expression vector suitable for expressing foreign genes in human embryonic stem cells has proven to be a non-trivial task. Most expression vectors otherwise useful in mammalian cells do not work at any reasonable degree of efficiency in human embryonic stem cells. It has been found here that there are two expression vectors which will permit the expression of foreign genes in human embryonic stem cells. The two vectors are an Epstein-Barr virus based expression vector and the second type is a Lentivirus expression vector.  
     [0024] The Epstein-Barr virus (EBV) expression vector is based on a commercially available expression vector. The EBV contains a genome of about 172 kb and is maintained in the transformed cells extrachromosomally as a multi-copy, circular episome. The episome replicates with the cells and is faithfully partitioned to daughter cells. It has been found that an EBV vector is capable of transferring into human embryonic stem cells an episome containing an inserted DNA construct which is then faithfully expressed in the transformed stem cells. Further information about the EBV based expression vectors is contained in attachment 1 included with this submission.  
     [0025] Lentivirus vectors are based on the family of retroviruses including human immunodeficiency virus (HIV). Lentivirus vectors have proven efficient at transforming human embryonic stem cells. The lentiviral genome contains the structural genes common to all retroviruses (gag, pol, and env) and in addition contain two regulatory (tat and rev) and four accessory genes (vpr, vif vpu, and nef). The four accessory genes function in replication and pathogenesis in vivo and can be eliminated from lentiviral vectors, although some of these may offer benefit for some cell types for the expression vector function. In the lentiviral vectors contemplated to be used in this process described in this invention, plasmid vectors are used to express gag, pol, tat and rev in a packaging cell line, but intact copies of the genes are eliminated from the actual transfer expression vector transferred into the stem cell lines. The tropism of retroviruses is largely determined by the env protein which binds to specific cell surface receptors. Therefore cells which lack the appropriate cell surface receptor may be difficult to transform with the retrovirus. To confer the broadest possible tropism on the expression vector, the lentiviral vectors will be pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein. VSV-G interacts directly with the phospholipid component of a cell membrane to mediate viral entry into the cell by fusion with the cell membrane. VSV-G can replace the env protein in retroviruses to produce hybrid pseudotype virus particles with extremely broad tropism. The expression vectors can be derived from vectors which contain cis-acting sequences of HIV required for packaging, reverse transcription, and integration. These sequences include the HIV 5′ LTR, the leader sequence and the 5′ splice donor site, about 360 base pairs of the gag gene, with a restriction endonuclease frame shift mutation preventing translation of gag sequences, 700 base pairs of the env gene containing the Rev-responsive element (RRE) for nuclear export, a 3′ splice acceptor site and the HIV 3′ LTR. To reduce the possible interfering effects of viral sequences on gene expression controlled by an internal promoter, all vectors will also contain a 400 base pair deletion of the U3 region of the 3′ HIV LTR. Because this sequence is copied to the 5′ LTR during a reverse transcription in the subsequent genomic integration, the 5′ LTR promoter/enhancer is rendered non-functional after integration into the host genome. Vectors with this modification are sometimes referred to as self-inactivating.  
     [0026] Lentiviral vectors can infect non-dividing cells, an important attribute for this purpose. The ability of lentiviral vectors to infect non-dividing cells is mediated through the gene products of the gag, pol, and vpr genes. Recently it has been identified that another element involved in nuclear import is a cis-determinant present within the pol coding region, known as the central purine tract (cPPT). For this reason, the cPPT region will be incorporated into the lentiviral vectors for use in this invention. Integration position effects due to the random integration of the retrovirus into the genome contribute to transcriptional silencing of vectors shortly after integration and also contribute to expression variegation and extinction of expression.  
     [0027] To construct an actual lentivirus vector, we began with a gift lentivirus vector, pSIN-EF-EGFP from Robert Hawley, American Red Cross, Rockville, Md. To modify the vector for use with human embryonic stem cells, the vector was modified to decrease the size of the construct and to make recombinations more efficient. To simplify subsequent cloning steps, the GFP cassette was removed from the vector by digestion with BamHI and religating the vector, the relegated vector being designated pSIN-EF-del. To decrease the size of the vector, 1909 base pairs were deleted from the NcoI site (8990) to the HpaI site (202) to make a vector named pSIN-EF-del2). This decreased the vector size from 10659 to 8750 base pairs. Then the GATEWAY (Invitrogen, Life Sciences, Carlsbad, Calif.) vector conversion cassette B was added to the SmaI site at base pair 4082 in the original vector to make a vector designated pSIN-EF-del2-GATEWAY. This vector can now be used to directly transfer individual clones, groups of clones, or entire libraries to the lentivirus vector for use in over-expression studies. The vector is illustrated in FIG. 1 and the sequence of the vector is contained in SEQ:ID:NO:1.  
     [0028] The development of an EBV-based vector has been carried forward. An EBV vector was acquired from the laboratory of William Sugden, the University of Wisconsin. To modify the vector as received (p2300) for use with human embryonic stem cells, the promoter was changed from the CMV promoter to the EF1 alpha promoter. In addition, a polylinker was added to make the vector easier to manipulate with conventional ligation-mediated cloning procedures. In addition, the GATEWAY cassette was added to the vector to make the vector compatible with the recombination system.  
     [0029] To make these changes, we began by amplifying the gene for green fluorescent protein (GFP) using primers JS 1 and JS2 below, which contain multiple restriction sites. The PCR product was digested with NotI and ClaI and cloned into NotI/ClaI sites of p2300. The resulting plasmid was designated pJMS001. Next the EF1 alpha promoter was amplified with the primers JS5 and JS7 below, the product was digested with SmaI and EcoRI, and the resulting fragment was ligated into the NruI/EcoRI site of pJMS001. The resulting plasmid was named pJMS002. Then pJMS002 was cut with EcoRi and BamHI, and the ends were blunted using T4-DNA polymerase. GATEWAY cassette B was ligated into the plasmid, resulting in a plasmid named pJMS002-GATEWAY. This vector, adapted for use in the method described here, is illustrated in FIG. 2 and its sequence is set forth in SEQ:ID:NO:2.  
                          Primer list           JS1:       GCATCGATTTCGAAGAATTCCACCGGTCGCCACCATGGTG               JS2:       AAAAGGAAAAGCGGCCGCCTCGAGGGATCCTTTACTTGTACAGCTCGTCC               JS5:       CGGCCCGGGGTGAGGCTCCGGTGCCCGTC               JS7:       GGCGAATTCGAACTCGAGACCACGTGTTCACGACACC          
 
     [0030] 
    
     
       
         1 
         
           
             2  
           
           
             1  
             10463  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            1 

gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc     60 

acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta    120 

tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc caaacttccc    180 

accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta aacctgtgat    240 

tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac tacaaactta    300 

gtagttggaa gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta    360 

ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata    420 

tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga    480 

ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga    540 

cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc    600 

ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact    660 

ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct    720 

cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga    780 

tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct    840 

tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat    900 

ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt    960 

gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg   1020 

cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg   1080 

ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg   1140 

atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt   1200 

atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga   1260 

aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact   1320 

tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa   1380 

agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc   1440 

acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa   1500 

gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg   1560 

caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg   1620 

ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg   1680 

ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg   1740 

cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc   1800 

tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa   1860 

aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac   1920 

agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct   1980 

taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat   2040 

tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt   2100 

atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg   2160 

tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc   2220 

tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag   2280 

acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgccac aaatggcagt   2340 

attcatccac aattttaaaa gaaagggggg gattgggggg tacagtgcag gggaaagaat   2400 

agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat   2460 

tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggatcg ataagctttg   2520 

caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc   2580 

ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca   2640 

gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc   2700 

ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga   2760 

gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc   2820 

agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc   2880 

ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt   2940 

cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt   3000 

gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt   3060 

cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct   3120 

gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt   3180 

atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg   3240 

gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc   3300 

cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg   3360 

ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca   3420 

gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa   3480 

aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg   3540 

ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg   3600 

gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca   3660 

gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc   3720 

attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagga   3780 

attcgatatc aagcttatcg atagatctgt cgactaaatt ctgcagtcga cggtaccgcg   3840 

ggatcaacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat   3900 

atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc   3960 

cagtcactat ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt   4020 

ataatgtgtg gattttgagt taggatccgg cgagattttc aggagctaag gaagctaaaa   4080 

tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac   4140 

attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata   4200 

ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc   4260 

acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg   4320 

agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa   4380 

cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt   4440 

cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga   4500 

atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg   4560 

ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg   4620 

acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg   4680 

tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa   4740 

gatctggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt   4800 

gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtgtgctat   4860 

gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat   4920 

gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt   4980 

gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa   5040 

atgaacggct cttttgctga cgagaacagg gactggtgaa atgcagttta aggtttacac   5100 

ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac   5160 

gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc   5220 

ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga   5280 

tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga   5340 

aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct   5400 

tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt   5460 

atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg   5520 

tttctcgttc agctttcttg tacaaagtgg ttgatcccgg gatccctcga gacctagaaa   5580 

aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag   5640 

aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa   5700 

tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag   5760 

ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt   5820 

tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc   5880 

aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta   5940 

actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat   6000 

gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc   6060 

cttgacatta taatagattt agcaggaatt gaactaggag tggagcacac aggcaaagct   6120 

gcagaagtac ttggaagaag ccaccagaga tactcacgat tctgcacata cctggctaat   6180 

cccagatcct aaggattaca ttaagtttac taacatttat ataatgattt atagtttaaa   6240 

gtataaactt atctaattta ctattctgac agatattaat taatcctcaa atatcataag   6300 

agatgattac tattatcccc atttaacaca agaggaaact gagagggaaa gatgttgaag   6360 

taattttccc acaattacag catccgttag ttacgactct atgatcttct gacacaaatt   6420 

ccatttactc ctcaccctat gactcagtcg aatatatcaa agttatggac attatgctaa   6480 

gtaacaaatt acccttttat atagtaaata ctgagtagat tgagagaaga aattgtttgc   6540 

aaacctgaat agcttcaaga agaagagaag tgaggataag aataacagtt gtcatttaac   6600 

aagttttaac aagtaacttg gttagaaagg gattcaaatg cataaagcaa gggataaatt   6660 

tttctggcaa caagactata caatataacc ttaaatatga cttcaaataa ttgttggaac   6720 

ttgataaaac taattaaata ttattgaaga ttatcaatat tataaatgta atttactttt   6780 

aaaaagggaa catagaaatg tgtatcatta gagtagaaaa caatccttat tatcacaatt   6840 

tgtcaaaaca agtttgttat taacacaagt agaatactgc attcaattaa gttgactgca   6900 

gattttgtgt tttgttaaaa ttagaaagag ataacaacaa tttgaattat tgaaagtaac   6960 

atgtaaatag ttctacatac gttcttttga catcttgttc aatcattgat cgaagttctt   7020 

tatcttggaa gaatttgttc caaagactct gaaataagga aaacaatcta ttatatagtc   7080 

tcacaccttt gttttacttt tagtgatttc aatttaataa tgtaaatggt taaaatttat   7140 

tcttctctga gatcatttca cattgcagat agaaaacctg agactggggt aatttttatt   7200 

aaaatctaat ttaatctcag aaacacatct ttattctaac atcaattttt ccagtttgat   7260 

attatcatat aaagtcagcc ttcctcatct gcaggttcca caacaaaaat ccaaccaact   7320 

gtggatcaaa aatattggga aaaaattaaa aatagcaata caacaataaa aaaatacaaa   7380 

tcagaaaaac agcacagtat aacaacttta tttagcattt acaatctatt aggtattata   7440 

agtaatctag aattaattcc gtgtattcta tagtgtcacc taaatcgtat gtgtatgata   7500 

cataaggtta tgtattaatt gtagccgcgt tctaacgaca atatgtacaa gcctaattgt   7560 

gtagcatctg gcttactgaa gcagacccta tcatctctct cgtaaactgc cgtcagagtc   7620 

ggtttggttg gacgaacctt ctgagtttct ggtaacgccg tcccgcaccc ggaaatggtc   7680 

agcgaaccaa tcagcagggt catcgctagc cagatcctct acgccggacg catcgtggcc   7740 

ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg   7800 

gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca   7860 

ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg   7920 

gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag   7980 

ggagagcgtc gaatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca   8040 

gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc   8100 

cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc   8160 

atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt   8220 

catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac   8280 

ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc   8340 

ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt   8400 

cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct   8460 

ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga   8520 

tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag   8580 

cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca   8640 

actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga   8700 

aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag   8760 

tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc   8820 

ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa   8880 

tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt   8940 

gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg   9000 

gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt   9060 

tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg   9120 

gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat   9180 

ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact   9240 

gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa   9300 

aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt   9360 

ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt   9420 

ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg   9480 

tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca   9540 

gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt   9600 

agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga   9660 

taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc   9720 

gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact   9780 

gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga   9840 

caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg   9900 

aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt   9960 

tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt  10020 

acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga  10080 

ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac  10140 

gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc  10200 

tctccccgcg cgttggccga ttcattaatg cagctgtgga atgtgtgtca gttagggtgt  10260 

ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca  10320 

gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat  10380 

ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg  10440 

cccagttccg cccattctcc gcc                                          10463 

 
           
             2  
             9249  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            2 

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg     60 

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg    120 

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc    180 

ttagggttag gcgttttgcg ctgcttcggg ggtgaggctc cggtgcccgt cgtgaggctc    240 

cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg    300 

ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt    360 

cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt    420 

cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg    480 

tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc    540 

cacctggctc cagtacgtga ttcttgatcc cgagctggag ccaggggcgg gccttgcgct    600 

ttaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc    660 

gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt    720 

taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg    780 

ggccaggatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt    840 

gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga    900 

cgggggtcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc    960 

cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag   1020 

cggaaagatg gccgcttccc ggccctgctc cagggggctc aaaatggagg acgcggcgct   1080 

cgggagagcg ggcgggtgag tcacccacac aaaggaaagg ggcctttccg tcctcagccg   1140 

tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctgga   1200 

gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc   1260 

acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg   1320 

aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa   1380 

gtttttttct tccatttcag gtgtcaagaa cacatggtct cgagttcatc aacaagtttg   1440 

tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt   1500 

ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg   1560 

ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt   1620 

tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca   1680 

ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc   1740 

agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa   1800 

agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc   1860 

tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg   1920 

atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct   1980 

ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt   2040 

gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct   2100 

cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact   2160 

tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc   2220 

cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta   2280 

atgaattaca acagtactgc gatgagtggc agggcggggc gtaaagatct ggatccggct   2340 

tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat   2400 

atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac   2460 

agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc   2520 

ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa   2580 

gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt   2640 

gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag   2700 

ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat   2760 

ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc   2820 

ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc   2880 

ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa   2940 

cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc   3000 

tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt   3060 

tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt   3120 

tcttgtacaa agtggttgat tccctcgagg cggccgcggg cgccagtgtg ctggaattaa   3180 

ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag cgggcatgac   3240 

ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc   3300 

ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct ttttgttgtc   3360 

aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa tgacatccac   3420 

tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga gcatgcatct   3480 

agggcggcca attccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct   3540 

tggaataagg ccggtgtgcg tttgtctata tgtgattttc caccatattg ccgtcttttg   3600 

gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt   3660 

cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg   3720 

aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac   3780 

ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg   3840 

cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct   3900 

caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg   3960 

atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag   4020 

gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa gcttgccaca   4080 

acccacaagg agacgacctt ccatgaccga gtacaagccc acggtgcgcc tcgccacccg   4140 

cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac   4200 

gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt   4260 

cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt   4320 

ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc   4380 

gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct   4440 

ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga   4500 

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg   4560 

cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg   4620 

gctcggcttc accgtcaccg ccgacgtcga gtgcccgaag gaccgcgcga cctggtgcat   4680 

gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag   4740 

cgcacgaccc catggctccg accgaagccg acccgggcgg ccccgccgac cccgcacccg   4800 

cccccgaggc ccaccgactc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc   4860 

cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc   4920 

actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct   4980 

attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg   5040 

catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg   5100 

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac   5160 

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgcctgg   5220 

ccggggtccc ccggaaactc ggccgtggtg accatgcagg aaaaggacaa gcagcgaaaa   5280 

ttcacgcccc cttgggaggt ggcggcatat gcaaaggata gcactcccac tctactactg   5340 

ggtatcatat gctgactgta tatgcatgag gatagcatat gctacccgga tacagattag   5400 

gatagcatat actacccaga tatagattag gatagcatat gctacccaga tatagattag   5460 

gatagcctat gctacccaga tataaattag gatagcatat actacccaga tatagattag   5520 

gatagcatat gctacccaga tatagattag gatagcctat gctacccaga tatagattag   5580 

gatagcatat gctacccaga tatagattag gatagcatat gctatccaga tatttgggta   5640 

gtatatgcta cccagatata aattaggata gcatatacta ccctaatctc tattaggata   5700 

gcatatgcta cccggataca gattaggata gcatatacta cccagatata gattaggata   5760 

gcatatgcta cccagatata gattaggata gcctatgcta cccagatata aattaggata   5820 

gcatatacta cccagatata gattaggata gcatatgcta cccagatata gattaggata   5880 

gcctatgcta cccagatata gattaggata gcatatgcta tccagatatt tgggtagtat   5940 

atgctaccca tggcaacatt agcccaccgt gctctcagcg acctcgtgaa tatgaggacc   6000 

aacaaccctg tgcttggcgc tcaggcgcaa gtgtgtgtaa tttgtcctcc agatcgcagc   6060 

aatcgcgccc ctatcttggc ccgcccacct acttatgcag gtattccccg gggtgccatt   6120 

agtggttttg tgggcaagtg gtttgaccgc agtggttagc ggggttacaa tcagccaagt   6180 

tattacaccc ttattttaca gtccaaaacc gcagggcggc gtgtgggggc tgacgcgtgc   6240 

ccccactcca caatttcaaa aaaaagagtg gccacttgtc tttgtttatg ggccccattg   6300 

gcgtggagcc ccgtttaatt ttcgggggtg ttagagacaa ccagtggagt ccgctgctgt   6360 

cggcgtccac tctctttccc cttgttacaa atagagtgta acaacatggt tcacctgtct   6420 

tggtccctgc ctgggacaca tcttaataac cccagtatca tattgcacta ggattatgtg   6480 

ttgcccatag ccataaattc gtgtgagatg gacatccagt ctttacggct tgtccccacc   6540 

ccatggattt ctattgttaa agatattcag aatgtttcat tcctacacta gtatttattg   6600 

cccaaggggt ttgtgagggt tatattggtg tcatagcaca atgccaccac tgaacccccc   6660 

gtccaaattt tattctgggg gcgtcacctg aaaccttgtt ttcgagcacc tcacatacac   6720 

cttactgttc acaactcagc agttattcta ttagctaaac gaaggagaat gaagaagcag   6780 

gcgaagattc aggagagttc actgcccgct ccttgatctt cagccactgc ccttgtgact   6840 

aaaatggttc actaccctcg tggaatcctg accccatgta aataaaaccg tgacagctca   6900 

tggggtggga gatatcgctg ttccttagga cccttttact aaccctaatt cgatagcata   6960 

tgcttcccgt tgggtaacat atgctattga attagggtta gtctggatag tatatactac   7020 

tacccgggaa gcatatgcta cccgtttagg gttataccgt cgacctctag ctagagcttg   7080 

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac   7140 

aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc   7200 

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg   7260 

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct   7320 

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac   7380 

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga   7440 

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat   7500 

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac   7560 

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct   7620 

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg   7680 

ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg   7740 

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt   7800 

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg   7860 

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac   7920 

ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga   7980 

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt   8040 

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt   8100 

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga   8160 

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc   8220 

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct   8280 

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata   8340 

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca   8400 

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga   8460 

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga   8520 

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg   8580 

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga   8640 

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt   8700 

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct   8760 

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca   8820 

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat   8880 

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga   8940 

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc   9000 

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg   9060 

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc   9120 

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt   9180 

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca   9240 

cctgacgtc                                                           9249