LOCUS Exported 10864 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid encoding the Cas9 gene and a gRNA expression cassette allowing to clone a single or multiple gRNAs for DSBs induction in yeast. Constructed by Aubin Fleiss. ACCESSION . VERSION . KEYWORDS pAEF5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10864) TITLE Yeast Protocols 4th Edition--A Versatile Protocol to Generate Translocations in Yeast Genomes using CRISPR/Cas9 JOURNAL Unpublished REFERENCE 2 (bases 1 to 10864) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10864 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 260..282 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(320..338) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 379..1721 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 711..758 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1748..1852 /gene="bla" /label=AmpR promoter CDS 1853..2713 /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(2071..2090) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2884..3472 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3373..3392 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3626..3643 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3760..3781 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3796..3826 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3834..3850 /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 3839..3861 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3858..3874 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3858..3874 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3893..3913 /label=T3 /note="T3 promoter, forward primer" promoter 3895..3913 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3931..4329 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" primer_bind 4333..4349 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 4333..4349 /label=pBluescriptSK /note="For pBluescript vector" CDS 4361..8464 /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 8477..8497 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 8513..8760 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(8529..8547) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter 8782..9050 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 9071..9145 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 9150..9169 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" primer_bind complement(9191..9210) /label=T7 /note="T7 promoter, forward primer" promoter complement(9192..9210) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9220..9237) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9220..9236) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9229..9251) /label=M13/pUC Forward /note="In lacZ gene" gene 9341..52 /label=hphMX6 /note="yeast selectable marker conferring hygromycin resistance" promoter 9341..9684 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 9685..10713 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" terminator 10719..52 /label=TEF terminator /note="Ashbya gossypii TEF terminator" ORIGIN 1 gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat 61 tcgatactaa cgccgccatc cagtgtcgaa aacgagctcg aattcatcga tgatatcaga 121 tccactagtg gcctatgcgg cccaatctgc tctgatgccg catagttaag ccagccccga 181 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 241 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 301 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 361 ataatggttt cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact 421 aatccaattg aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata 481 cgataccccg catggaatgg gataatatca caggaggtac tagactacct ttcatcctac 541 ataaatagac gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca 601 tgtatatata tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg 661 tgcgcagctc gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta 721 ttccgaagtt cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa 781 gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa 841 aatattgcga ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc 901 tgtgctatat ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa 961 ctcgacctct acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta 1021 gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg 1081 tagtatatag agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat 1141 caacgctatc actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc 1201 ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg 1261 gaagtggagt caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta 1321 accttaacgg acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag 1381 gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt 1441 gtagaacaaa aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt 1501 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca 1561 tttttgtttt acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg 1621 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg 1681 ttgcattttt gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg 1741 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 1801 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 1861 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 1921 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1981 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 2041 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 2101 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 2161 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 2221 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 2281 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 2341 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 2401 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 2461 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 2521 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 2581 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 2641 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 2701 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 2761 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 2821 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2881 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2941 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3001 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3061 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3121 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3181 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3241 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 3301 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 3361 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 3421 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 3481 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 3541 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 3601 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg agagacgccc 3661 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 3721 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 3781 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 3841 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa 3901 ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct actccttttt 3961 tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca 4021 cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag 4081 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa 4141 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg 4201 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 4261 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 4321 taatctaagt tttctagaac tagtggatcc cccgggaaaa atggacaaga agtactccat 4381 tgggctcgat atcggcacaa acagcgtcgg ttgggccgtc attacggacg agtacaaggt 4441 gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct 4501 cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac 4561 agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag 4621 taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt 4681 ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc 4741 gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga 4801 taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca 4861 cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca 4921 actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga 4981 cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc 5041 acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg 5101 gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag 5161 caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc 5221 agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg 5281 agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga 5341 gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta 5401 caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc 5461 aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga 5521 ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa 5581 tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga 5641 ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg 5701 gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg 5761 caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc 5821 tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt 5881 gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt 5941 caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc 6001 tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga 6061 ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt 6121 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct 6181 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga 6241 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt 6301 catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat 6361 caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg 6421 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga 6481 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct 6541 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact 6601 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa 6661 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg 6721 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca 6781 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga 6841 actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct 6901 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag 6961 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct 7021 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg 7081 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat 7141 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga 7201 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag 7261 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc 7321 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga 7381 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca 7441 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa 7501 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg 7561 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct 7621 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa 7681 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga 7741 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc 7801 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac 7861 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata 7921 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga 7981 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc 8041 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg 8101 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga 8161 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct 8221 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga 8281 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt 8341 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact 8401 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg 8461 agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg agtcatgtaa 8521 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa 8581 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 8641 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt 8701 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc 8761 ggccggtacc gcgggcccgc ttctttgaaa agataatgta tgattatgct ttcactcata 8821 tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg 8881 aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt 8941 cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg 9001 tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc agaagagcca 9061 tggctcttca gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 9121 ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctg cggccgcggt 9181 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg 9241 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccagatc 9301 tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac 9361 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat 9421 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc 9481 gcgaagcaaa aattacggct cctcgctgcg gacctgcgag cagggaaacg ctcccctcac 9541 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt 9601 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca 9661 catcacatcc gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc 9721 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 9781 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 9841 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 9901 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 9961 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 10021 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 10081 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 10141 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 10201 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 10261 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 10321 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 10381 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 10441 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 10501 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 10561 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 10621 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 10681 cgccccagca ctcgtccgag ggcaaaggaa taatcagtac tgacaataaa aagattcttg 10741 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa 10801 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa 10861 gtgc //