LOCUS Exported 9174 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pX459 V2.0 (Plasmid #62988) with the K848A, K1003A, and R1060A mutations. ACCESSION . VERSION . KEYWORDS pX459V2.0-eSpCas9(1.1) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9174) AUTHORS Tomoko Kato-Inui, Gou Takahashi, Szuyin Hsu, Yuichiro Miyaoka TITLE Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair JOURNAL Nucleic Acids Research REFERENCE 2 (bases 1 to 9174) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9174 /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..1232 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1244..1253 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1253..1318 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1325..1345 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1370..5470 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Slaymaker et al., 2016)" /label=eSpCas9(1.1) /note="carries the mutations K848A, K1003A, and R1060A" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5471..5518 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5534..5587 /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 5588..6187 /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(5588..5607) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6084..6104 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(6215..6232) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6221..6428 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6437..6577 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6437..6566 /label=AAV2 ITR rep_origin 6652..7107 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6739..6758) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6949..6970 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7124..7143) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7243..7265 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7303..7321) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7389..7493 /gene="bla" /label=AmpR promoter CDS 7494..8354 /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(7712..7731) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8525..9113 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9014..9033 /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 ggggcggggg ggggcggggg gggggggggg gcggggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct 1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga 1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc 1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa 1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat 1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta 1381 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta 1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa 1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa 1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat 1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt 1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga 1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag 1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg 1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt 1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg 1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct 2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag 2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca 2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca 2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat 2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata 2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga 2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg 2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg 2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt 2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg 2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac 2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat 2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg 2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga 2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac 2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa 3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa 3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga 3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga 3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact 3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga 3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa 3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc 3421 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa 3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc 3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga 3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag 3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga 3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca 3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga 3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag 3901 ctttctggcc gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg 3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca 4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag 4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg 4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga 4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga 4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca 4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctgcgctgga 4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag 4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt 4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aaggcgcctc tgatcgagac 4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa 4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt 4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga 4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt 4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg 4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa 4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga 4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga 5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct 5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta 5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc 5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca 5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa 5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc 5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct 5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga 5521 attcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc 5581 tggcccaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag 5641 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga 5701 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg 5761 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac 5821 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt 5881 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc 5941 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg 6001 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc 6061 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt 6121 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg 6181 tgcctgagaa ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag 6241 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 6301 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 6361 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat 6421 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct 6481 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 6541 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc 6601 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct 6661 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 6721 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 6781 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 6841 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct 6901 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 6961 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt 7021 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 7081 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg 7141 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 7201 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 7261 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 7321 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 7381 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 7441 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 7501 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 7561 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 7621 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 7681 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 7741 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 7801 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 7861 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 7921 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 7981 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 8041 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 8101 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 8161 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta 8221 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 8281 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 8341 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 8401 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 8461 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 8521 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 8581 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 8641 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 8701 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 8761 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 8821 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 8881 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 8941 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 9001 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 9061 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 9121 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt //