LOCUS Exported 10899 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of SpCas9 and sgRNA. ACCESSION . VERSION . KEYWORDS KA2058_pX330A-G2P SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10899) AUTHORS Adachi K, Kopp W, Wu G, Heising S, Greber B, Stehling M, Arauzo-Bravo MJ, Boerno ST, Timmermann B, Vingron M, Scholer HR TITLE Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive Pluripotency. JOURNAL Cell Stem Cell. 2018 Aug 2;23(2):266-275.e6. doi: 10.1016/j.stem.2018.05.020. Epub 2018 Jun 14. PUBMED 29910149 REFERENCE 2 (bases 1 to 10899) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10899 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 440..725 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1004 /label=chicken beta-actin promoter intron 1005..1233 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1245..1254 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1254..1319 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1326..1346 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1371..5471 /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 5472..5519 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5547..5564) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5553..5760 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5769..5909 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5769..5898 /label=AAV2 ITR promoter 6054..6265 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 6278..6546 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 6575..6584 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6605..7267 /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" CDS 7337..7390 /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 7391..7990 /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(7391..7410) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7887..7907 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(8044..8064) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 8106..8227 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8143..8162) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8197..8216 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 8377..8832 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8464..8483) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8674..8695 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8849..8868) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8968..8990 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(9028..9046) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 9114..9218 /gene="bla" /label=AmpR promoter CDS 9219..10079 /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(9437..9456) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10250..10838 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10739..10758 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1381 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5521 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5581 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5641 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5701 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5761 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5821 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5881 gagcgagcga gcgcgcagct gcctgcagga aagagcccac aacccctcac ttggtgggcc 5941 agtcctctga tagactgtgt cccctggata cccgtattct agagctagcg aattcgaatt 6001 taaatcggat ccgcggccgc gaaggatctg cgatcgctcc ggtgcccgtc agtgggcaga 6061 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc 6121 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 6181 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 6241 caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg 6301 cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc 6361 cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag 6421 accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct 6481 ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta 6541 cagatccaag ctgtgaccgg cgcctacgct agacgccacc atggagagcg acgagagcgg 6601 cctgcccgcc atggagatcg agtgccgcat caccggcacc ctgaacggcg tggagttcga 6661 gctggtgggc ggcggagagg gcacccccaa gcagggccgc atgaccaaca agatgaagag 6721 caccaaaggc gccctgacct tcagccccta cctgctgagc cacgtgatgg gctacggctt 6781 ctaccacttc ggcacctacc ccagcggcta cgagaacccc ttcctgcacg ccatcaacaa 6841 cggcggctac accaacaccc gcatcgagaa gtacgaggac ggcggcgtgc tgcacgtgag 6901 cttcagctac cgctacgagg ccggccgcgt gatcggcgac ttcaaggtgg tgggcaccgg 6961 cttccccgag gacagcgtga tcttcaccga caagatcatc cgcagcaacg ccaccgtgga 7021 gcacctgcac cccatgggcg ataacgtgct ggtgggcagc ttcgcccgca ccttcagcct 7081 gcgcgacggc ggctactaca gcttcgtggt ggacagccac atgcacttca agagcgccat 7141 ccaccccagc atcctgcaga acgggggccc catgttcgcc ttccgccgcg tggaggagct 7201 gcacagcaac accgagctgg gcatcgtgga gtaccagcac gccttcaaga cccccatcgc 7261 cttcgccaga tcccgcgctc agtcgtccaa ttctgccgtg gacggcaccg ccggacccgg 7321 ctccaccgga tctcgcgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa 7381 tcccggccct atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 7441 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 7501 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 7561 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 7621 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 7681 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg 7741 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa 7801 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc 7861 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 7921 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 7981 cggtgcctga aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 8041 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgcgtt 8101 aactaaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 8161 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 8221 tatcttatca tgtctggaat tgactcaaat gatgtcaatt agtctatcag aagctatctg 8281 gtctcccttc cgggcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc 8341 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 8401 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 8461 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 8521 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 8581 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 8641 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 8701 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 8761 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 8821 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 8881 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 8941 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 9001 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 9061 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 9121 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 9181 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 9241 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 9301 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 9361 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 9421 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 9481 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 9541 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 9601 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 9661 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 9721 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 9781 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 9841 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 9901 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg 9961 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 10021 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 10081 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 10141 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 10201 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 10261 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 10321 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 10381 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 10441 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 10501 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 10561 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 10621 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 10681 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 10741 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 10801 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 10861 tacggttcct ggccttttgc tggccttttg ctcacatgt //