LOCUS       sequence-292507-       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: epiCRISPR
FEATURES             Location/Qualifiers
     source          1..14029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..297
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     49..69
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     220..239
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        318..393
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    441..558
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        627..838
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      858..867
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             867..890
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             897..917
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             942..5042
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             942..5042
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5043..5090
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5106..5162
                     /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             5163..5759
                     /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(5163..5182)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5659..5679
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5787..5840
                     /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             5871..6533
                     /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    6653..6787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6677..6696)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6731..6750
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6930..6952)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      7512..9301
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     CDS             complement(9603..11528)
                     /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
                     AGGGAGAGGGAGGXGGXGGXGXGGGXGGGGGXGGXGGXGGGGGGGGGGGXGGGGGGGGX
                     GGXGGXGXGGGAGXGGGAGGXGXGGGAGGXGGXGXGGGAGAGGAGGAGGXGXGGXGXGG
                     GXGGXGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGXGGXGGXGXGGGXGGXGXG
                     GGXGGXGXGGGGGGGGGGXGGXGXGGXGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRG
                     GSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPP
                     PGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGR
                     RKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSL
                     YNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLK
                     DAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE
                     GEEGQE"
     primer_bind     complement(11963..11981)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12049..12153
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12154..12222,12223..13014)
                     /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(12372..12391)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13185..13773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13674..13693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(13962..13982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 cattatggta cctttcagac ccacctccca accccgaggg gacccagaga gggcctattt
       61 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
      121 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      181 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      241 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgag
      301 aagagcgatg ctcttcggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
      361 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat cttgagacaa
      421 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
      481 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
      541 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
      601 ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
      661 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
      721 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
      781 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
      841 gtcgtgacgc gggatccgcc accatggatt acaaagacga tgacgataag atggccccaa
      901 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
      961 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     1021 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     1081 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     1141 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     1201 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1261 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1321 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1381 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1441 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1501 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     1561 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     1621 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     1681 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     1741 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     1801 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     1861 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     1921 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     1981 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     2041 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2101 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2161 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2221 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2281 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2341 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2401 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2461 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     2521 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     2581 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     2641 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     2701 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     2761 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     2821 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     2881 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     2941 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3001 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3061 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3121 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3181 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3241 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3301 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3361 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3421 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     3481 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     3541 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     3601 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     3661 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     3721 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     3781 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     3841 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     3901 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     3961 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     4021 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4081 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     4141 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4201 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4261 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4321 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     4381 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     4441 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     4501 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     4561 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     4621 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     4681 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     4741 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     4801 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     4861 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     4921 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     4981 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     5041 acaagcgtcc tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa gctagcggca
     5101 gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc
     5161 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     5221 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     5281 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     5341 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     5401 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     5461 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     5521 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     5581 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     5641 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     5701 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg
     5761 cgatcgccga aggatccgcg gccgctgagg gcagaggaag tcttctaaca tgcggtgacg
     5821 tggaggagaa tcccggccct tccggaatgg agagcgacga gagcggcctg cccgccatgg
     5881 agatcgagtg ccgcatcacc ggcaccctga acggcgtgga gttcgagctg gtgggcggcg
     5941 gagagggcac ccccaagcag ggccgcatga ccaacaagat gaaaagcacc aaaggcgccc
     6001 tgaccttcag cccctacctg ctgagccacg tgatgggcta cggcttctac cacttcggca
     6061 cctaccccag cggctacgag aaccccttcc tgcacgccat caacaacggc ggctacacca
     6121 acacccgcat cgagaagtac gaggacggcg gcgtgctgca cgtgagcttc agctaccgct
     6181 acgaggccgg ccgcgtgatc ggcgacttca aggtggtggg caccggcttc cccgaggaca
     6241 gcgtgatctt caccgacaag atcatccgca gcaacgccac cgtggagcac ctgcacccca
     6301 tgggcgataa cgtgctggtg ggcagcttcg cccgcacctt cagcctgcgc gacggcggct
     6361 actacagctt cgtggtggac agccacatgc acttcaagag cgccatccac cccagcatcc
     6421 tgcagaacgg gggccccatg ttcgccttcc gccgcgtgga ggagctgcac agcaacaccg
     6481 agctgggcat cgtggagtac cagcacgcct tcaagacccc catcgccttc gccagatccc
     6541 gcgctcagtc gtccaattct gccgtggacg gcaccgccgg acccggctcc accggatctc
     6601 gctagagctg aatctaagtc gacttaagaa ccgctcgagg ccggcaaggc cggatccaga
     6661 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     6721 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     6781 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     6841 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atccggctgc
     6901 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     6961 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     7021 gttggcgggt gtcggggcgc agccatgagg tcgactctag aggatcgatc cccgccccgg
     7081 acgaactaaa cctgactacg acatctctgc cccttcttcg cggggcagtg catgtaatcc
     7141 cttcagttgg ttggtacaac ttgccaactg ggccctgttc cacatgtgac acgggggggg
     7201 accaaacaca aaggggttct ctgactgtag ttgacatcct tataaatgga tgtgcacatt
     7261 tgccaacact gagtggcttt catcctggag cagactttgc agtctgtgga ctgcaacaca
     7321 acattgcctt tatgtgtaac tcttggctga agctcttaca ccaatgctgg gggacatgta
     7381 cctcccaggg gcccaggaag actacgggag gctacaccaa cgtcaatcag aggggcctgt
     7441 gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat aaggccccct
     7501 tgttaaccct aaacgggtag catatgcttc ccgggtagta gtatatacta tccagactaa
     7561 ccctaattca atagcatatg ttacccaacg ggaagcatat gctatcgaat tagggttagt
     7621 aaaagggtcc taaggaacag cgatatctcc caccccatga gctgtcacgg ttttatttac
     7681 atggggtcag gattccacga gggtagtgaa ccattttagt cacaagggca gtggctgaag
     7741 atcaaggagc gggcagtgaa ctctcctgaa tcttcgcctg cttcttcatt ctccttcgtt
     7801 tagctaatag aataactgct gagttgtgaa cagtaaggtg tatgtgaggt gctcgaaaac
     7861 aaggtttcag gtgacgcccc cagaataaaa tttggacggg gggttcagtg gtggcattgt
     7921 gctatgacac caatataacc ctcacaaacc ccttgggcaa taaatactag tgtaggaatg
     7981 aaacattctg aatatcttta acaatagaaa tccatggggt ggggacaagc cgtaaagact
     8041 ggatgtccat ctcacacgaa tttatggcta tgggcaacac ataatcctag tgcaatatga
     8101 tactggggtt attaagatgt gtcccaggca gggaccaaga caggtgaacc atgttgttac
     8161 actctatttg taacaagggg aaagagagtg gacgccgaca gcagcggact ccactggttg
     8221 tctctaacac ccccgaaaat taaacggggc tccacgccaa tggggcccat aaacaaagac
     8281 aagtggccac tctttttttt gaaattgtgg agtgggggca cgcgtcagcc cccacacgcc
     8341 gccctgcggt tttggactgt aaaataaggg tgtaataact tggctgattg taaccccgct
     8401 aaccactgcg gtcaaaccac ttgcccacaa aaccactaat ggcaccccgg ggaatacctg
     8461 cataagtagg tgggcgggcc aagatagggg cgcgattgct gcgatctgga ggacaaatta
     8521 cacacacttg cgcctgagcg ccaagcacag ggttgttggt cctcatattc acgaggtcgc
     8581 tgagagcacg gtgggctaat gttgccatgg gtagcatata ctacccaaat atctggatag
     8641 catatgctat cctaatctat atctgggtag cataggctat cctaatctat atctgggtag
     8701 tatatgctat cctaatctgt atccgggtag catatgctat cctaatagag attagggtag
     8761 catatgctat cctaatctat atctgggtag cataggctat cctaatctat atctgggtag
     8821 tatatgctat cctaatctgt atccgggtag catatgctat cctaatagag attagggtag
     8881 tatatgctat cctaatttat atctgggtag catatactac ccaaatatct ggatagcata
     8941 tgctatccta atctatatct gggtagcata tgctatccta atctatatct gggtagcata
     9001 ggctatccta atctatatct gggtagcata tgctatccta atctatatct gggtagtata
     9061 tgctatccta atttatatct gggtagcata ggctatccta atctatatct gggtagcata
     9121 tgctatccta atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata
     9181 tgctatcctc atgcatatac agtcagcata tgatacccag tagtagagtg ggagtgctat
     9241 cctttgcata tgccgccacc tcccaagggg gcgtgaattt tcgctgcttg tccttttcct
     9301 gctggttgct cccattctta ggtgaattta aggaggccag gctaaagccg tcgcatgtct
     9361 gattgctcac caggtaaatg tcgctaatgt tttccaacgc gagaaggtgt tgagcgcgga
     9421 gctgagtgac gtgacaacat gggtatgccc aattgcccca tgttgggagg acgaaaatgg
     9481 tgacaagaca gatggccaga aatacaccaa cagcacgcat gatgtctact ggggatttat
     9541 tctttagtgc gggggaatac acggctttta atacgattga gggcgtctcc taacaagtta
     9601 catcactcct gcccttcctc accctcatct ccatcacctc cttcatctcc gtcatctccg
     9661 tcatcaccct ccgcggcagc cccttccacc ataggtggaa accagggagg caaatctact
     9721 ccatcgtcaa agctgcacac agtcaccctg atattgcagg taggagcggg ctttgtcata
     9781 acaaggtcct taatcgcatc cttcaaaacc tcagcaaata tatgagtttg taaaaagacc
     9841 atgaaataac agacaatgga ctcccttagc gggccaggtt gtgggccggg tccaggggcc
     9901 attccaaagg ggagacgact caatggtgta agacgacatt gtggaatagc aagggcagtt
     9961 cctcgcctta ggttgtaaag ggaggtctta ctacctccat atacgaacac accggcgacc
    10021 caagttcctt cgtcggtagt cctttctacg tgactcctag ccaggagagc tcttaaacct
    10081 tctgcaatgt tctcaaattt cgggttggaa cctccttgac cacgatgctt tccaaaccac
    10141 cctccttttt tgcgcctgcc tccatcaccc tgaccccggg gtccagtgct tgggccttct
    10201 cctgggtcat ctgcggggcc ctgctctatc gctcccgggg gcacgtcagg ctcaccatct
    10261 gggccacctt cttggtggta ttcaaaataa tcggcttccc ctacagggtg gaaaaatggc
    10321 cttctacctg gagggggcct gcgcggtgga gacccggatg atgatgactg actactggga
    10381 ctcctgggcc tcttttctcc acgtccacga cctctccccc tggctctttc acgacttccc
    10441 cccctggctc tttcacgtcc tctaccccgg cggcctccac tacctcctcg accccggcct
    10501 ccactacctc ctcgaccccg gcctccactg cctcctcgac cccggcctcc acctcctgct
    10561 cctgcccctc ctgctcctgc ccctcctcct gctcctgccc ctcctscccc tcctsctcct
    10621 scccctccys cccctccycc tcctccccct cctccccctc ctsctcctsc ccctcctscc
    10681 cctcctccts ctcctscccc tcctscccct cctcctsctc ctscccctcc tscccctcct
    10741 sctcctgccc ctcctgcccc tcctgctcct gcccctcctg cccctcctgc tcctgcccct
    10801 cctgctcctg cccctcctgc tcctgcccct cctgctcctg cccctcctsc ccctcctscc
    10861 cctcctccts ctcctscccc tcctsctcct kcccctcctg cccctcctgc ccctcctgct
    10921 cctgcccctc ctcctsctcc tscccctcct scccctcctg cccctcctcc tsctcctscc
    10981 cctcctgccc ctcctcctsc tcctgcccct cctcctsctc ctscccctcc tscccctcct
    11041 scccctcctc ctcctcctcc ccctcctscc cctcctcctc ctcctccccc tcctcctcct
    11101 cctscccctc ctscycctcc tscccctcct cctcctccts cccctcctcc tsctcctscc
    11161 cctcctsccc ctcctscccc tcctgcccct cctcctgctc ctgcccctcc tcctgctcct
    11221 gcccctcctg ctcctgcccc tcccgctcct gctcctgctc ctgttccacc gtgggtccct
    11281 ttgcagccaa tgcaacttgg acgtttttgg ggtctccgga caccatctct atgtcttggc
    11341 cctgatcctg agccgcccgg ggctcctggt cttccgcctc ctcgtcctcg tcctcttccc
    11401 cgtcctcgtc catggttatc accccctctt ctttgaggtc cactgccgcc ggagccttct
    11461 ggtccagatg tgtctccctt ctctcctagg ccatttccag gtcctgtacc tggcccctcg
    11521 tcagacatga ttcacactaa aagagatcaa tagacatctt tattagacga cgctcagtga
    11581 atacagggag tgcagactcc tgccccctcc aacagccccc ccaccctcat ccccttcatg
    11641 gtcgctgtca gacagatcca ggtctgaaaa ttccccatcc tccgaaccat cctcgtcctc
    11701 atcaccaatt actcgcagcc cggaaaactc ccgctgaaca tcctcaagat ttgcgtcctg
    11761 agcctcaagc caggcctcaa attcctcgtc cccctttttg ctggacggta gggatgggga
    11821 ttctcgggac ccctcctctt cctcttcaag gtcaccagac agagatgcta ctggggcaac
    11881 ggaagaaaag ctgggtgcgg cctgtgagga tcagcttatc gatgataagc tgtcaaacat
    11941 gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
    12001 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
    12061 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
    12121 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
    12181 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
    12241 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
    12301 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
    12361 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
    12421 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
    12481 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
    12541 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
    12601 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
    12661 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
    12721 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
    12781 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
    12841 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
    12901 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
    12961 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
    13021 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
    13081 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
    13141 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
    13201 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
    13261 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
    13321 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
    13381 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
    13441 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
    13501 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
    13561 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
    13621 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
    13681 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
    13741 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
    13801 ttcctggcct tttgctggcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
    13861 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
    13921 aaggccatcc agcctcgcgt cgaactagat gatccggctg tggaatgtgt gtcagttagg
    13981 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatg
//
