LOCUS Exported 11034 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION p426_Cas9_gRNA-ARS805a without ribozyme All-in-one CRISPR plasmid for integrating, deleting, or replacing a gene in S. cerevisiae. High-copy, URA3 plasmid contains human codon-optimized Cas9 and a guide RNA targeting ARS805a sequence TTATTTGAATGATATTTAGT in yeast chromosome 8.. ACCESSION . VERSION . KEYWORDS p426_Cas9_gRNA-R-ARS805a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11034) AUTHORS Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A TITLE A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. JOURNAL Nucleic Acids Res. 2017 Jan 9;45(1):496-508. doi: 10.1093/nar/gkw1023. Epub 2016 Nov 28. PUBMED 27899650 REFERENCE 2 (bases 1 to 11034) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11034 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 63..82 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 316..333 /label=L4440 /note="L4440 vector, forward primer" protein_bind 450..471 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 486..516 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 524..540 /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 529..551 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 548..564 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 548..564 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 583..603 /label=T3 /note="T3 promoter, forward primer" promoter 585..603 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_RNA 758..833 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 919..1623 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 1633..5736 /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 FLVEEDKKNERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIYLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5749..5769 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 5785..6032 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6050..6069) /label=T7 /note="T7 promoter, forward primer" promoter complement(6051..6069) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6079..6096) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6079..6095) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6088..6110) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6236..6691 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6323..6342) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6533..6554 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6822..7625) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" promoter complement(7626..7841) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(7865..7884) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7984..8006 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8044..8062) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 8103..9445 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 8435..8482 /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 9472..9576 /gene="bla" /label=AmpR promoter CDS 9577..10437 /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(9795..9814) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10608..162 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 61 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 481 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 601 agggaacaaa agctggagct agtatactct ttcttcaaca attaaatact ctcggtagcc 661 aagttggttt aaggcgcaag actgtaattt atcactacga aatcttgaga tcgggcgttc 721 gactcgcccc cgggagatta tttgaatgat atttagtgtt ttagagctag aaatagcaag 781 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 841 attttttgtc actattgtta tgtaaaatgc cacctctgac agtatggaac gcaaacttct 901 gtctagtgga tagtcgacat ccttttgttg tttccgggtg tacaatatgg acttcctctt 961 ttctggcaac caaacccata catcgggatt cctataatac cttcgttggt ctccctaaca 1021 tgtaggtggc ggaggggaga tatacaatag aacagatacc agacaagaca taatgggcta 1081 aacaagacta caccaattac actgcctcat tgatggtggt acataacgaa ctaatactgt 1141 agccctagac ttgatagcca tcatcatatc gaagtttcac tacccttttt ccatttgcca 1201 tctattgaag taataatagg cgcatgcaac ttcttttctt tttttttctt ttctctctcc 1261 cccgttgttg tctcaccata tccgcaatga caaaaaaatg atggaagaca ctaaaggaaa 1321 aaattaacga caaagacagc accaacagat gtcgttgttc cagagctgat gaggggtatc 1381 tcgaagcaca cgaaactttt tccttccttc attcacgcac actactctct aatgagcaac 1441 ggtatacggc cttccttcca gttacttgaa tttgaaataa aaaaaagttt gctgtcttgc 1501 tatcaagtat aaatagacct gcaattatta atcttttgtt tcctcgtcat tgttctcgtt 1561 ccctttcttc cttgtttctt tttctgcaca atatttcaag ctataccaag catacaatca 1621 acttggaaaa aaatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc 1681 ggttgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc 1741 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg 1801 gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag 1861 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct 1921 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagaa cgagcgccac 1981 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat 2041 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc 2101 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca 2161 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc 2221 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg 2281 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc 2341 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc 2401 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac 2461 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg 2521 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg 2581 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag 2641 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa 2701 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt 2761 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa 2821 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg 2881 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac 2941 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc 3001 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg 3061 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact 3121 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag 3181 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag 3241 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac 3301 cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac 3361 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat 3421 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt 3481 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg 3541 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat 3601 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga 3661 aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt catgcagttg 3721 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag 3781 ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga 3841 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc 3901 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac 3961 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt 4021 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg 4081 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac 4141 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg 4201 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc 4261 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag 4321 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc 4381 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat 4441 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt 4501 actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga 4561 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca 4621 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac 4681 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac 4741 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag 4801 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag 4861 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa 4921 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc 4981 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct 5041 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa 5101 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa 5161 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt 5221 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt 5281 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg 5341 tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag 5401 ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc 5461 tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag 5521 cacagggata agcccatcag ggagcaggca gaaaacatta tctacttgtt tactctgacc 5581 aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac 5641 acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat 5701 gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag 5761 aggaaggtgt gatctcttct cgagtcatgt aattagttat gtcacgctta cgttcacgcc 5821 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc 5881 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc 5941 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga 6001 aggttttggg acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga 6061 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 6121 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 6181 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg 6241 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 6301 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 6361 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 6421 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 6481 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 6541 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 6601 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 6661 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc 6721 tgtgcggtat ttcacaccgc atagggtaat aactgatata attaaattga agctctaatt 6781 tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat 6841 cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc 6901 cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca 6961 tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg 7021 ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata 7081 aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct 7141 ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc 7201 tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg 7261 tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat 7321 ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg 7381 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc 7441 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc 7501 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta 7561 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc 7621 gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact 7681 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc 7741 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac 7801 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta tggtgcactc 7861 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7921 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7981 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 8041 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttagt 8101 atgatccaat atcaaaggaa atgatagcat tgaaggatga gactaatcca attgaggagt 8161 ggcagcatat agaacagcta aagggtagtg ctgaaggaag catacgatac cccgcatgga 8221 atgggataat atcacaggag gtactagact acctttcatc ctacataaat agacgcatat 8281 aagtacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata tatatataca 8341 ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca gctcgcgttg 8401 cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga agttcctatt 8461 ctctagaaag tataggaact tcagagcgct tttgaaaacc aaaagcgctc tgaagacgca 8521 ctttcaaaaa accaaaaacg caccggactg taacgagcta ctaaaatatt gcgaataccg 8581 cttccacaaa cattgctcaa aagtatctct ttgctatata tctctgtgct atatccctat 8641 ataacctacc catccacctt tcgctccttg aacttgcatc taaactcgac ctctacattt 8701 tttatgttta tctctagtat tactctttag acaaaaaaat tgtagtaaga actattcata 8761 gagtgaatcg aaaacaatac gaaaatgtaa acatttccta tacgtagtat atagagacaa 8821 aatagaagaa accgttcata attttctgac caatgaagaa tcatcaacgc tatcactttc 8881 tgttcacaaa gtatgcgcaa tccacatcgg tatagaatat aatcggggat gcctttatct 8941 tgaaaaaatg cacccgcagc ttcgctagta atcagtaaac gcgggaagtg gagtcaggct 9001 ttttttatgg aagagaaaat agacaccaaa gtagccttct tctaacctta acggacctac 9061 agtgcaaaaa gttatcaaga gactgcatta tagagcgcac aaaggagaaa aaaagtaatc 9121 taagatgctt tgttagaaaa atagcgctct cgggatgcat ttttgtagaa caaaaaagaa 9181 gtatagattc tttgttggta aaatagcgct ctcgcgttgc atttctgttc tgtaaaaatg 9241 cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg ttttacaaaa 9301 atgaagcaca gattcttcgt tggtaaaata gcgctttcgc gttgcatttc tgttctgtaa 9361 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgttcta 9421 caaaatgaag cacagatgct tcgttcaggt ggcacttttc ggggaaatgt gcgcggaacc 9481 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 9541 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 9601 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 9661 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 9721 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 9781 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 9841 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 9901 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 9961 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 10021 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 10081 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 10141 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 10201 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 10261 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 10321 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 10381 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 10441 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 10501 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 10561 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 10621 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 10681 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 10741 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 10801 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 10861 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 10921 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 10981 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaag //