LOCUS Exported 9838 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Cas9 protein, under the control of the TEF1 promoter, and CYC terminator. KanMX resistance. ACCESSION . VERSION . KEYWORDS pCfB2312 (TEF1p-Cas9-CYC1t_kanMX) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9838) AUTHORS Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I TITLE EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. JOURNAL Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. Epub 2016 Jun 23. PUBMED 27166612 REFERENCE 2 (bases 1 to 9838) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9838 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 59..562 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 599..703 /gene="bla" /label=AmpR promoter CDS 704..1564 /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(922..941) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1735..2323 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2224..2243 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2477..2494 /label=L4440 /note="L4440 vector, forward primer" promoter 2647..2677 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2685..2701 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 2690..2712 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2709..2725 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2709..2725 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2744..2764 /label=T3 /note="T3 promoter, forward primer" promoter 2746..2764 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 2782..3180 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" primer_bind 3184..3200 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 3184..3200 /label=pBluescriptSK /note="For pBluescript vector" CDS 3212..7315 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7328..7348 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7364..7611 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(7380..7398) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(7629..7648) /label=T7 /note="T7 promoter, forward primer" promoter complement(7630..7648) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(7658..7675) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7658..7674) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7667..7689) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7819..8199 /direction=RIGHT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7906..7925) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8116..8137 /label=F1ori-F /note="F1 origin, forward primer" protein_bind 8251..8284 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." gene complement(8347..9703) /label=kanMX /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" terminator 8347..8544 /label=TEF terminator /note="Ashbya gossypii TEF terminator" CDS complement(8550..9359) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 9273..9292 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(9360..9703) /label=TEF promoter /note="Ashbya gossypii TEF promoter" protein_bind 9758..9791 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ORIGIN 1 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttaggacgga 61 tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa tttgggaatt 121 tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta aataaagaag 181 gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa 241 aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag atatacattc 301 gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac 361 aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat 421 ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta ttttttcttt 481 aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa tttaaattat 541 aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg gaaatgtgcg 601 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 661 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 721 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 781 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 841 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 901 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 961 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 1021 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 1081 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 1141 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 1201 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 1261 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 1321 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 1381 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 1441 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 1501 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 1561 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 1621 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 1681 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 1741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 1801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 1861 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 1921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 1981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 2041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 2101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 2161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 2221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 2281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 2341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 2401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 2461 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 2521 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 2581 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc attaggcacc 2641 ccaggcttta cactttatgc ttccggctcc tatgttgtgt ggaattgtga gcggataaca 2701 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta 2761 aagggaacaa aagctggagc tcatagcttc aaaatgtttc tactcctttt ttactcttcc 2821 agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact 2881 aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa 2941 agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat 3001 cacgtttctt tttcttgaaa attttttttt tgattttttt ctctttcgat gacctcccat 3061 tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt 3121 ctattacaac tttttttact tcttgctcat tagaaagaaa gcatagcaat ctaatctaag 3181 ttttctagaa ctagtggatc ccccgggaaa aatggacaag aagtactcca ttgggctcga 3241 tatcggcaca aacagcgtcg gttgggccgt cattacggac gagtacaagg tgccgagcaa 3301 aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc 3361 cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg 3421 cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat 3481 ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga 3541 taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga 3601 aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga 3661 cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat 3721 cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca 3781 gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc 3841 aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc 3901 tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc 3961 caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac 4021 ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt 4081 tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac 4141 ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca 4201 agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat 4261 tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga 4321 ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct 4381 ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat 4441 cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta 4501 cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta 4561 ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga 4621 agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc 4681 cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa 4741 acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt 4801 cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga 4861 cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa 4921 aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc 4981 cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga 5041 ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga 5101 gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca 5161 gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat 5221 ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa 5281 ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa 5341 agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag 5401 cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt 5461 aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac 5521 ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga 5581 actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa 5641 gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat 5701 caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc tcaaagatga 5761 ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt 5821 cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa 5881 actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga 5941 gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca 6001 cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat 6061 tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt 6121 tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa 6181 tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta 6241 cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg 6301 caaggccacc gctaagtact tcttttacag caatattatg aattttttca agaccgagat 6361 tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg 6421 agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc 6481 gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat 6541 cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa 6601 atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga 6661 gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga 6721 gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt 6781 caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg 6841 gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc 6901 taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga 6961 agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat 7021 cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt 7081 gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat 7141 ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac 7201 catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca 7261 gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag 7321 ggctgacccc aagaagaaga ggaaggtgtg atctcttctc gagtcatgta attagttatg 7381 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga 7441 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 7501 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 7561 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccggtac 7621 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc 7681 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 7741 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 7801 tgaatggcga atggcgcgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 7861 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 7921 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 7981 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 8041 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 8101 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 8161 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aattgggtgc 8221 ataggccact agtggatctg atatcaccta ataacttcgt atagcataca ttatacgaag 8281 ttatattaag ggttctcgag agctcgtttt cgacactgga tggcggcgtt agtatcgaat 8341 cgacagcagt atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc 8401 acttaacttc gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac 8461 atttgattaa aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa 8521 aacaagaatc tttttattgt cagtactgat tagaaaaact catcgagcat caaatgaaac 8581 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat 8641 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg 8701 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta 8761 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc 8821 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca 8881 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg 8941 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca 9001 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttgccg 9061 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc 9121 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg 9181 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat 9241 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa 9301 tcagcatcca tgttggaatt taatcgcggc ctcgaaacgt gagtcttttc cttacccatg 9361 gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta aaaggaagta 9421 tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac aggggcgcgg 9481 cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg 9541 aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt 9601 atacatgggg atgtatgggc taaatgtacg ggcgacagtc acatcatgcc cctgagctgc 9661 gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctggcc gggtgacccg 9721 gcggggacga ggcaagctaa acagatctct agacctaata acttcgtata gcatacatta 9781 tacgaagtta tattaagggt tgtcgacctg cagcgtacga agcttcagct gacgcgat //