LOCUS       sequence-287960-       14029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14029)
  AUTHORS   Xie Y, Wang D, Lan F, Wei G, Ni T, Chai R, Liu D, Hu S, Li M, Li D, 
            Wang H, Wang Y
  TITLE     An episomal vector-based CRISPR/Cas9 system for highly efficient 
            gene knockout in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2017 May 24;7(1):2320. doi: 10.1038/s41598-017-02456-y.
  PUBMED    28539611
REFERENCE   2  (bases 1 to 14029)
  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"; journalName: "Sci Rep."; 
            date: "2017-05-24"; volume: "7(1):2320. doi"; pages: " 
            10.1038/s41598-017-02456-y"
COMMENT     Sequence Label: epiCRISPRn
FEATURES             Location/Qualifiers
     source          1..14029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    393..510
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        579..790
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      810..819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             819..842
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             849..869
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(894..917,918..920,921..4994)
                     /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             894..4994
                     /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             4995..5042
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5058..5114
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5115..5711
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5115..5134)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5611..5631
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5739..5792
                     /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             5823..6485
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
     polyA_signal    6605..6739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6629..6648)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6683..6702
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6882..6904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      7464..9253
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     CDS             complement(9555..11480)
                     /codon_start=1
                     /product="Epstein-Barr nuclear antigen 1, also known as 
                     EBNA-1"
                     /label=EBNA1
                     /note="crucial for latent viral infection, and for episomal
                     amplification of vectors containing the oriP origin (Okita 
                     et al., 2013)"
                     /translation="MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGR
                     GRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAG
                     AGGGAGAGGGAGGAGGAGGAGAGGGAGXGGGAGGXGGAGAGGGAGXGGGXGGAGXGGGX
                     GGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGXGAGGAGAGG
                     GAGGAGGAGAGGXGXGGXGXGGXGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
                     GGGGGGGGGGGGGGGGGGGGGGGGGGXGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRG
                     GSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPP
                     PGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGR
                     RKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSL
                     YNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLK
                     DAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE
                     GEEGQE"
     primer_bind     complement(11915..11933)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12001..12105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12106..12174,12175..12966)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(12324..12343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13137..13725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13626..13645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(13914..13934)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg agaagagcga tgctcttcgg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattctag
      361 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
      421 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
      481 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
      541 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
      601 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
      661 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
      721 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
      781 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata
      841 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
      901 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
      961 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1021 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1081 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1141 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1201 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1261 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1321 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1381 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1441 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1501 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1561 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     1621 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     1681 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     1741 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     1801 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     1861 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     1921 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     1981 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2041 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2101 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2161 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2221 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2281 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2341 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2401 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2461 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2521 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     2581 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     2641 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     2701 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     2761 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     2821 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     2881 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     2941 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3001 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3061 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3121 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3181 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3241 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3301 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3361 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3421 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3481 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3541 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     3601 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     3661 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     3721 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     3781 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     3841 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     3901 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     3961 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4021 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4081 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4141 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4201 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4261 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4321 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4381 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4441 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4501 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4561 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     4621 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     4681 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     4741 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     4801 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     4861 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     4921 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     4981 agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga
     5041 aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg
     5101 agaaccccgg ccccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     5161 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5221 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5281 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5341 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5401 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5461 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     5521 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5581 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5641 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5701 agcccggtgc cgcgatcgcc gaaggatccg cggccgctga gggcagagga agtcttctaa
     5761 catgcggtga cgtggaggag aatcccggcc cttccggaat ggagagcgac gagagcggcc
     5821 tgcccgccat ggagatcgag tgccgcatca ccggcaccct gaacggcgtg gagttcgagc
     5881 tggtgggcgg cggagagggc acccccaagc agggccgcat gaccaacaag atgaaaagca
     5941 ccaaaggcgc cctgaccttc agcccctacc tgctgagcca cgtgatgggc tacggcttct
     6001 accacttcgg cacctacccc agcggctacg agaacccctt cctgcacgcc atcaacaacg
     6061 gcggctacac caacacccgc atcgagaagt acgaggacgg cggcgtgctg cacgtgagct
     6121 tcagctaccg ctacgaggcc ggccgcgtga tcggcgactt caaggtggtg ggcaccggct
     6181 tccccgagga cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc accgtggagc
     6241 acctgcaccc catgggcgat aacgtgctgg tgggcagctt cgcccgcacc ttcagcctgc
     6301 gcgacggcgg ctactacagc ttcgtggtgg acagccacat gcacttcaag agcgccatcc
     6361 accccagcat cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg gaggagctgc
     6421 acagcaacac cgagctgggc atcgtggagt accagcacgc cttcaagacc cccatcgcct
     6481 tcgccagatc ccgcgctcag tcgtccaatt ctgccgtgga cggcaccgcc ggacccggct
     6541 ccaccggatc tcgctagagc tgaatctaag tcgacttaag aaccgctcga ggccggcaag
     6601 gccggatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     6661 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     6721 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     6781 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     6841 tgatccggct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     6901 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6961 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga ggtcgactct agaggatcga
     7021 tccccgcccc ggacgaacta aacctgacta cgacatctct gccccttctt cgcggggcag
     7081 tgcatgtaat cccttcagtt ggttggtaca acttgccaac tgggccctgt tccacatgtg
     7141 acacgggggg ggaccaaaca caaaggggtt ctctgactgt agttgacatc cttataaatg
     7201 gatgtgcaca tttgccaaca ctgagtggct ttcatcctgg agcagacttt gcagtctgtg
     7261 gactgcaaca caacattgcc tttatgtgta actcttggct gaagctctta caccaatgct
     7321 gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc
     7381 agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt
     7441 ataaggcccc cttgttaacc ctaaacgggt agcatatgct tcccgggtag tagtatatac
     7501 tatccagact aaccctaatt caatagcata tgttacccaa cgggaagcat atgctatcga
     7561 attagggtta gtaaaagggt cctaaggaac agcgatatct cccaccccat gagctgtcac
     7621 ggttttattt acatggggtc aggattccac gagggtagtg aaccatttta gtcacaaggg
     7681 cagtggctga agatcaagga gcgggcagtg aactctcctg aatcttcgcc tgcttcttca
     7741 ttctccttcg tttagctaat agaataactg ctgagttgtg aacagtaagg tgtatgtgag
     7801 gtgctcgaaa acaaggtttc aggtgacgcc cccagaataa aatttggacg gggggttcag
     7861 tggtggcatt gtgctatgac accaatataa ccctcacaaa ccccttgggc aataaatact
     7921 agtgtaggaa tgaaacattc tgaatatctt taacaataga aatccatggg gtggggacaa
     7981 gccgtaaaga ctggatgtcc atctcacacg aatttatggc tatgggcaac acataatcct
     8041 agtgcaatat gatactgggg ttattaagat gtgtcccagg cagggaccaa gacaggtgaa
     8101 ccatgttgtt acactctatt tgtaacaagg ggaaagagag tggacgccga cagcagcgga
     8161 ctccactggt tgtctctaac acccccgaaa attaaacggg gctccacgcc aatggggccc
     8221 ataaacaaag acaagtggcc actctttttt ttgaaattgt ggagtggggg cacgcgtcag
     8281 cccccacacg ccgccctgcg gttttggact gtaaaataag ggtgtaataa cttggctgat
     8341 tgtaaccccg ctaaccactg cggtcaaacc acttgcccac aaaaccacta atggcacccc
     8401 ggggaatacc tgcataagta ggtgggcggg ccaagatagg ggcgcgattg ctgcgatctg
     8461 gaggacaaat tacacacact tgcgcctgag cgccaagcac agggttgttg gtcctcatat
     8521 tcacgaggtc gctgagagca cggtgggcta atgttgccat gggtagcata tactacccaa
     8581 atatctggat agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
     8641 atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct atcctaatag
     8701 agattagggt agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
     8761 atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct atcctaatag
     8821 agattagggt agtatatgct atcctaattt atatctgggt agcatatact acccaaatat
     8881 ctggatagca tatgctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     8941 ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     9001 ctgggtagta tatgctatcc taatttatat ctgggtagca taggctatcc taatctatat
     9061 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatctgtat
     9121 ccgggtagca tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag
     9181 tgggagtgct atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct
     9241 tgtccttttc ctgctggttg ctcccattct taggtgaatt taaggaggcc aggctaaagc
     9301 cgtcgcatgt ctgattgctc accaggtaaa tgtcgctaat gttttccaac gcgagaaggt
     9361 gttgagcgcg gagctgagtg acgtgacaac atgggtatgc ccaattgccc catgttggga
     9421 ggacgaaaat ggtgacaaga cagatggcca gaaatacacc aacagcacgc atgatgtcta
     9481 ctggggattt attctttagt gcgggggaat acacggcttt taatacgatt gagggcgtct
     9541 cctaacaagt tacatcactc ctgcccttcc tcaccctcat ctccatcacc tccttcatct
     9601 ccgtcatctc cgtcatcacc ctccgcggca gccccttcca ccataggtgg aaaccaggga
     9661 ggcaaatcta ctccatcgtc aaagctgcac acagtcaccc tgatattgca ggtaggagcg
     9721 ggctttgtca taacaaggtc cttaatcgca tccttcaaaa cctcagcaaa tatatgagtt
     9781 tgtaaaaaga ccatgaaata acagacaatg gactccctta gcgggccagg ttgtgggccg
     9841 ggtccagggg ccattccaaa ggggagacga ctcaatggtg taagacgaca ttgtggaata
     9901 gcaagggcag ttcctcgcct taggttgtaa agggaggtct tactacctcc atatacgaac
     9961 acaccggcga cccaagttcc ttcgtcggta gtcctttcta cgtgactcct agccaggaga
    10021 gctcttaaac cttctgcaat gttctcaaat ttcgggttgg aacctccttg accacgatgc
    10081 tttccaaacc accctccttt tttgcgcctg cctccatcac cctgaccccg gggtccagtg
    10141 cttgggcctt ctcctgggtc atctgcgggg ccctgctcta tcgctcccgg gggcacgtca
    10201 ggctcaccat ctgggccacc ttcttggtgg tattcaaaat aatcggcttc ccctacaggg
    10261 tggaaaaatg gccttctacc tggagggggc ctgcgcggtg gagacccgga tgatgatgac
    10321 tgactactgg gactcctggg cctcttttct ccacgtccac gacctctccc cctggctctt
    10381 tcacgacttc cccccctggc tctttcacgt cctctacccc ggcggcctcc actacctcct
    10441 cgaccccggc ctccactacc tcctcgaccc cggcctccac tgcctcctcg accccggcct
    10501 ccacctcctg ctcctgcccc tcctgctcct gcccctcctc ctgctcctgc ccctcctscc
    10561 cctcctcctc ctccccctcc tccccctcct cctccycccc ctcctccccc tccycctcct
    10621 ccccctcctc cccctccycc tcctcctccc cctcctcccc ctcctcctcc tcctccccct
    10681 cctccccctc ctcctcctcc ccctcctccc cctcctcctc ctccccctcc tccccctcct
    10741 cctcctsccc ctcctsctcc tscccctcct sctcctsccc ctcctgctcc tgcccctcct
    10801 gcccctcctg cccctcctcc tgctcctgcc cctcctgctc ctscccctcc tgcccctcct
    10861 gcccctcctg ctcctgcccc tcctcctgct cctgcycctc ctgcccctcc tgcccctcct
    10921 cctgctcctg cccctcctgc ccctcctcct gctcctgccc ctcctcctgc tcctgcccct
    10981 cctgcccctc ctscccctcc tcctsctcct gcccctccts cccctcctcc tsctcctgcc
    11041 cctcctcctg ctcctgcccc tcctscccct cctgcccctc ctcctsctcc tgcccctcct
    11101 cctgctcctg cccctcctgc ccctcctgcc cctcctgccc ctcctcctgc tcctgcccct
    11161 cctcctgctc ctgcccctcc tgctcctgcc cctcccgctc ctgctcctgc tcctgttcca
    11221 ccgtgggtcc ctttgcagcc aatgcaactt ggacgttttt ggggtctccg gacaccatct
    11281 ctatgtcttg gccctgatcc tgagccgccc ggggctcctg gtcttccgcc tcctcgtcct
    11341 cgtcctcttc cccgtcctcg tccatggtta tcaccccctc ttctttgagg tccactgccg
    11401 ccggagcctt ctggtccaga tgtgtctccc ttctctccta ggccatttcc aggtcctgta
    11461 cctggcccct cgtcagacat gattcacact aaaagagatc aatagacatc tttattagac
    11521 gacgctcagt gaatacaggg agtgcagact cctgccccct ccaacagccc ccccaccctc
    11581 atccccttca tggtcgctgt cagacagatc caggtctgaa aattccccat cctccgaacc
    11641 atcctcgtcc tcatcaccaa ttactcgcag cccggaaaac tcccgctgaa catcctcaag
    11701 atttgcgtcc tgagcctcaa gccaggcctc aaattcctcg tccccctttt tgctggacgg
    11761 tagggatggg gattctcggg acccctcctc ttcctcttca aggtcaccag acagagatgc
    11821 tactggggca acggaagaaa agctgggtgc ggcctgtgag gatcagctta tcgatgataa
    11881 gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
    11941 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
    12001 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
    12061 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
    12121 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
    12181 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
    12241 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
    12301 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
    12361 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
    12421 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
    12481 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
    12541 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
    12601 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
    12661 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
    12721 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
    12781 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
    12841 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
    12901 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
    12961 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
    13021 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
    13081 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
    13141 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
    13201 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
    13261 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
    13321 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
    13381 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
    13441 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
    13501 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
    13561 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
    13621 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
    13681 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
    13741 gcctttttac ggttcctggc cttttgctgg ccttgaagct gtccctgatg gtcgtcatct
    13801 acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga
    13861 atcataatgg ggaaggccat ccagcctcgc gtcgaactag atgatccggc tgtggaatgt
    13921 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
    13981 gcattatggt acctttcaga cccacctccc aaccccgagg ggacccaga
//
