LOCUS       sequence-175009-        9109 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CRISPR KO of Trp53.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9109)
  AUTHORS   Wang Q, Zou Y, Nowotschin S, Kim SY, Li QV, Soh CL, Su J, Zhang C, 
            Shu W, Xi Q, Huangfu D, Hadjantonakis AK, Massague J
  TITLE     The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal 
            Differentiation of Embryonic Stem Cells.
  JOURNAL   Cell Stem Cell. 2017 Jan 5;20(1):70-86. doi: 
            10.1016/j.stem.2016.10.002. Epub 2016 Nov 23.
  PUBMED    27889317
REFERENCE   2  (bases 1 to 9109)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2016.10.002"; journalName: "Cell Stem Cell"; date: 
            "2017-01-5- 5"; volume: "20"; issue: "1"; pages: "70-86"
COMMENT     Sequence Label: Px461-Cas9n-Trp53-sgRNA-beta
FEATURES             Location/Qualifiers
     source          1..9109
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      225..234
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             234..299
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             306..326
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(351..374,375..377,378..4451)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             351..4451
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4452..4499
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4515..4568
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4569..4571,4572..5282)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4569..4571,4572..5282)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4872..4891)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5219..5240
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5310..5327)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5316..5523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5532..5672
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5532..5661
                     /label=AAV2 ITR
     rep_origin      5747..6202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5834..5853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6044..6065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6219..6238)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6338..6360
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6398..6416)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6484..6588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6589..6657,6658..7449)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6807..6826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7620..8208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8109..8128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8270..8518
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8270..8290
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8441..8460
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8539..8614
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8711..8996
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
       61 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      121 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
      181 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
      241 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
      301 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
      361 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
      421 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
      481 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
      541 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
      601 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
      661 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
      721 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
      781 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
      841 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
      901 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
      961 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     1021 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
     1081 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     1141 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     1201 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     1261 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     1321 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     1381 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     1441 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     1501 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     1561 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     1621 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     1681 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     1741 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     1801 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     1861 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     1921 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     1981 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     2041 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     2101 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     2161 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     2221 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     2281 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     2341 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     2401 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     2461 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     2521 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     2581 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     2641 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     2701 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     2761 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     2821 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     2881 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     2941 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     3001 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     3061 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     3121 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     3181 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     3241 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     3301 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     3361 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     3421 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     3481 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     3541 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     3601 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     3661 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     3721 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     3781 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     3841 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     3901 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     3961 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     4021 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     4081 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     4141 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     4201 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     4261 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     4321 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     4381 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     4441 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     4501 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     4561 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     4621 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     4681 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     4741 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     4801 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     4861 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     4921 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     4981 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     5041 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     5101 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     5161 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     5221 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     5281 aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     5341 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     5401 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     5461 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     5521 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     5581 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     5641 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     5701 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     5761 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5821 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5881 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5941 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     6001 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6061 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     6121 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6181 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     6241 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     6301 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     6361 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     6421 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     6481 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6541 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     6601 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6661 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     6721 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     6781 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     6841 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     6901 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     6961 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     7021 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     7081 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     7141 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     7201 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     7261 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     7321 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     7381 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     7441 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     7501 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7741 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     7801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     8041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     8101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     8221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt
     8281 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     8341 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     8401 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     8461 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     8521 ggagctcctg acactcgggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     8581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc tagaaatagc
     8641 aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga caaatggctc
     8701 tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     8761 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     8821 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     8881 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
     8941 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     9001 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     9061 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcgggggg
//
