LOCUS Exported 19008 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Monocot Target-AID vector expressing rice-optimized nCas9-PmCDA1 with sgRNA targeting OsAls. ACCESSION . VERSION . KEYWORDS pMonAID_nCas9-PmCDA_Hyg_ALS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 19008) AUTHORS Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A TITLE Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. JOURNAL Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3833. PUBMED 28346401 REFERENCE 2 (bases 1 to 19008) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..19008 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 113..137 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" protein_bind 295..316 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 331..361 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 369..385 /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 374..396 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 393..409 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 393..409 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 485..509 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(559..575) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(560..576) /label=pBluescriptKS /note="For pBluescript vector" protein_bind 596..695 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" misc_RNA 1104..1179 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(1191..1215) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1221..1320) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 2111..2156 /label=minimal CaMV 35S promoter /note="minimal 35S promoter from cauliflower mosaic virus" 5'UTR 2167..2267 /label=OsADH 5'-UTR /note="translational enhancer from the 5'-UTR of the Oryza sativa alcohol dehydrogenase gene (Sugio et al., 2008)" CDS 2277..2297 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2322..6425 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6447..6467 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6660..6725 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 6732..7355 /codon_start=1 /product="cytidine deaminase from the sea lamprey Petromyzon marinus (Rogozin et al., 2007)" /label=PmCDA1 /translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV" CDS 7359..7379 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 9657..10002 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" CDS 10017..11042 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK K" protein_bind complement(12194..12318) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(12358..12375) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(12358..12374) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12367..12389) /label=M13/pUC Forward /note="In lacZ gene" misc_feature 12589..12613 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 13134..13925 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 14171..14759 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 14660..14679 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 14913..14930 /label=L4440 /note="L4440 vector, forward primer" misc_feature 14945..15085 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(15011..15030) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 15171..15193 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 15429..15623 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(15689..16762) /codon_start=1 /product="replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 RepA /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI LVMRYRNLIEGEASAGS" CDS complement(17191..17820) /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" ORIGIN 1 gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt 61 ttcacgccct tttaaatatc cgattattct aataaacgct cttttctctt aggtttaccc 121 gccaatatat cctgtcaaac actgatagtt taaactgaag gcgggaaacg acaatctgat 181 ccaagctcaa gctccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 241 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 301 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 361 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa 421 tttcaggtga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat 481 aatgccaact ttgtacaaaa aagcaggcgg cgcgtcctgc aggataggga taacagggta 541 atggcgcgcc aagcttatcg ataccgtcga cctcgagcgg ccgcgggaat tcgatcaaat 601 aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgatg agcaatgctt 661 ttttataatg ccaactttgt acaaaaaagc aggctctttt tttcttcttc ttcgttcata 721 cagttttttt ttgtttatca gcttacattt tcttgaaccg tagctttcgt tttcttcttt 781 ttaactttcc attcggagtt tttgtatctt gtttcatagt ttgtcccagg attagaatga 841 ttaggcatcg aaccttcaag aatttgattg aataaaacat cttcattctt aagatatgaa 901 gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca tttatatggg 961 aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct tcaaaagtcc 1021 cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga 1081 ttgcgcgtcc atggagatcc accgttttag agctagaaat agcaagttaa aataaggcta 1141 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctag acccagcttt 1201 cttgtacaaa gttggcatta acccagcttt cttgtacaaa gttggcatta taaaaaataa 1261 ttgctcatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg 1321 atcactagtg gatccgagct cggtacctta attaaattac cctgttatcc ctatcctgca 1381 ggattaacac ccaacttttc tatccaagct tgccaacatg gtggagcacg acactctcgt 1441 ctactccaag aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca 1501 acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat 1561 caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa 1621 ggctatcgtt caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 1681 gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 1741 acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 1801 accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 1861 attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 1921 aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 1981 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 2041 cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc 2101 actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagaggccgg 2161 tctagagaat tccaagcaac gaactgcgag tgattcaaga aaaaagaaaa cctgagcttt 2221 cgatctctac ggagtggttt cttgttcttt gaaaaagagg gggattacat atggctccta 2281 agaagaagcg gaaggttggt attcacgggg tgcctgcggc tatggacaag aagtactcga 2341 tcgggctggc catcggaaca aattctgtag gctgggctgt aataaccgat gagtacaagg 2401 tgccctctaa aaaatttaag gtccttggca atacggatag acattccata aagaagaatc 2461 ttatcggtgc gctgctcttt gacagcggcg agaccgcgga ggcgacccgg ttgaaacgca 2521 ccgcgagacg ccgttacaca aggcgtaaga atagaatctg ttatctccag gagatattct 2581 ctaatgaaat ggcgaaggta gacgattcct tctttcaccg tctggaggaa agttttctcg 2641 ttgaggaaga taagaaacat gaaagacacc cgatcttcgg aaacattgtc gacgaggttg 2701 cttatcatga aaagtaccct accatctacc atcttagaaa gaaacttgtt gacagcacgg 2761 ataaggctga tctcaggctg atatacctgg ctctggcaca tatgattaag ttcagagggc 2821 atttccttat cgaaggcgac ctgaatccag ataattcaga tgtagacaag ctcttcattc 2881 aacttgtgca gacttataat cagctcttcg aagaaaatcc aataaacgcg tcgggtgtag 2941 acgcaaaggc catactgtcc gctaggcttt ctaagtcacg tagacttgag aatctcattg 3001 cccaactccc cggcgagaag aagaacggct tgtttggaaa tctgatagcg ctgtccctgg 3061 gtcttacacc aaatttcaag agtaatttcg atttggcaga agatgctaag ttgcagctca 3121 gtaaagacac ctacgatgac gatcttgata atttgttggc tcagattggc gatcagtatg 3181 cagatctttt cttggccgct aagaatttgt ctgatgcaat tctgcttagc gacattttga 3241 gggttaatac agaaatcacc aaggcaccct tgtcggcgtc aatgataaag aggtatgatg 3301 agcaccacca agacctgacg ctcctcaagg ctcttgttcg gcagcaattg ccggagaagt 3361 acaaagagat cttcttcgac cagtctaaga acggatatgc gggctacata gacggtggag 3421 cgagtcagga ggaattctac aagttcataa agcccattct cgagaagatg gatggtacgg 3481 aagaactgct tgtgaaactt aacagagaag atcttttgcg gaagcagaga actttcgaca 3541 acggaagtat accacaccag atacatctcg gagagcttca tgctattctc agaagacaag 3601 aggatttcta ccctttcttg aaggataaca gagaaaagat agagaagatc ctcacgttta 3661 ggatccctta ctacgtaggt cctcttgctc gcggcaatag taggttcgcc tggatgaccc 3721 gcaagtctga agaaactatc accccttgga atttcgaaga ggttgtagac aaaggtgctt 3781 cagcacagag tttcattgag aggatgacca acttcgacaa gaacctcccc aacgaaaagg 3841 tcctgcctaa gcacagcctc ctctacgaat actttactgt ctataatgag cttacaaaag 3901 ttaagtacgt gacagaggga atgcggaagc ccgcattcct ttccggagaa caaaagaagg 3961 cgatcgtgga tcttctcttc aagacgaacc gcaaggtgac ggttaaacag ttgaaggaag 4021 attacttcaa gaagatagaa tgttttgata gcgtggaaat cagcggcgtc gaagataggt 4081 tcaacgcttc cctgggaacg taccacgatc tcctcaagat tatcaaagat aaggactttc 4141 ttgataacga agagaatgag gacatcttgg aagacattgt tctgacgctc accctgttcg 4201 aagatcgcga gatgattgag gaacgcttga agacctacgc acacctgttc gatgacaagg 4261 ttatgaagca acttaaacgg cgccggtata cgggctgggg acggctttcg cggaagctga 4321 taaatggaat ccgtgacaag cagtctggca agacaatact cgacttcttg aagtcggatg 4381 gttttgccaa tagaaatttt atgcaactca ttcatgatga ctcgcttact tttaaggagg 4441 acatccagaa ggcccaggta tcaggacagg gtgactcttt gcacgaacac atcgcgaacc 4501 tggcgggctc ccccgcgatt aagaagggaa ttttgcagac tgtcaaggtg gtcgatgaac 4561 tcgtgaaggt tatgggacgt cataagccgg aaaatattgt gattgagatg gctcgcgaga 4621 atcaaacaac acagaagggc caaaagaaca gtagagaacg catgaagcgc atcgaagagg 4681 gcatcaaaga gctgggcagt cagatcctta aagaacatcc agtcgagaat acacagcttc 4741 agaacgaaaa gctgtacctt tattaccttc aaaatgggcg tgatatgtat gtggatcagg 4801 aactcgatat caataggctg agtgactatg atgtcgacca tatcgtcccg caaagtttcc 4861 tcaaggacga cagtatagac aacaaagttc tcacacggtc agataagaat cgcggcaaga 4921 gcgataatgt accgtcggag gaggtagtca agaagatgaa gaattactgg cgccagttgc 4981 tcaacgccaa gctcatcact cagaggaaat ttgacaacct tacgaaagcc gagcggggcg 5041 gactctctga actggacaag gccggtttca taaagcgcca gctcgttgag acacgtcaaa 5101 ttactaagca cgtcgctcaa atattggatt cccgcatgaa tactaagtac gatgagaatg 5161 ataagctcat acgtgaagtt aaggtcatta ctctcaagtc caagcttgta tcggacttcc 5221 gtaaggactt ccaattctac aaggtccggg aaatcaataa ttatcaccat gcccatgacg 5281 cttatctgaa cgcggtcgtg ggcacggcac tcattaagaa atacccaaaa cttgagtcag 5341 aatttgttta cggggactat aaagtttatg acgtgcggaa gatgatagcg aagtcggaac 5401 aagagatagg aaaggcgact gcaaagtact ttttttactc caacataatg aatttcttta 5461 agaccgaaat aacccttgca aacggtgaaa tcagaaagcg gcctctgatt gaaacaaatg 5521 gcgagacggg cgagatcgtc tgggacaagg ggagggactt cgcaacggtt cgcaaggtcc 5581 ttagcatgcc gcaagtaaat atagttaaga agacggaagt tcagaccggc ggctttagta 5641 aagaaagcat acttcctaaa aggaattccg acaaactgat agcgcgcaag aaggactggg 5701 atccaaagaa gtatggagga tttgactccc caaccgttgc ttatagcgtg ttggtagtag 5761 ccaaggtgga aaagggtaag tctaagaaat tgaagtcggt gaaggagttg ttggggataa 5821 ctataatgga gcggagttcg ttcgagaaga acccaattga ctttctcgaa gccaaaggct 5881 acaaggaggt caagaaggac ctgattatta agttgccaaa gtactcgctc ttcgaactcg 5941 agaacgggag aaagcgtatg ctggcgtcgg cgggcgagct gcagaaagga aacgagctgg 6001 ctttgccatc gaaatacgta aatttcctgt acctcgcctc acattatgag aagcttaaag 6061 ggtctccaga agacaatgaa cagaagcagc tgtttgttga acagcacaag cactacttgg 6121 acgagattat agaacaaatc tccgagttct ctaaacgggt tatccttgca gacgccaatt 6181 tggataaggt cctctcggct tataataagc atagagataa gccaatccgg gagcaggctg 6241 aaaatatcat acacctcttt acgttgacta atttgggtgc gccagcggca ttcaagtact 6301 tcgatacaac aatcgatcgt aagcgctaca caagcactaa ggaagtcctg gacgcgacgc 6361 tgatacacca gtccattact ggactgtatg aaaccagaat agatcttagc cagctcggcg 6421 gtgatggagg agggcctagg gctgatccta agaagaagag gaaggttgga ggagggccag 6481 gagcagagta tgttagagcg ttgtttgatt ttaacggtaa cgatgaagag gatttaccct 6541 ttaagaaagg cgacattctc aggattaggg ataaacctga agagcaatgg tggaatgctg 6601 aggatagtga aggcaaacga ggaatgattt tagtgccgta tgtggagaaa tattcgggtg 6661 actacaaaga tcatgatggt gattacaaag accatgacat cgactacaag gatgatgatg 6721 ataagtcagg gatgacagat gctgaatatg tcagaatcca cgaaaagttg gacatttaca 6781 cgtttaagaa gcagttcttc aacaacaaga aatctgtttc gcataggtgc tatgtgcttt 6841 tcgaactaaa acgtcgtgga gaaagacggg cttgcttttg gggttacgcg gttaacaaac 6901 cacaatcagg tactgaacga ggaatacacg ctgaaatctt ttctatccga aaggttgagg 6961 aatatctacg tgacaatcct ggacagttca ctatcaattg gtattctagc tggtcaccat 7021 gtgcagattg tgctgagaag attctcgaat ggtacaatca agagcttaga ggcaatggac 7081 atacattgaa aatatgggca tgcaagctct actacgaaaa gaatgccaga aaccaaattg 7141 ggctttggaa cttgagggat aatggagttg ggcttaatgt catggtttct gagcactatc 7201 aatgttgtcg gaagatcttc atacaaagtt cccataacca gttgaatgag aacagatggt 7261 tagagaaaac ccttaaaaga gccgagaaga gaagatccga actgagcatt atgatacagg 7321 tcaaaattct gcataccact aagagtccag ctgtaggtcc taagaagaaa cgtaaagtag 7381 ggccctgatc caggcctccc agctttcgtc cgtatcatcg gtttcgacaa cgttcgtcaa 7441 gttcaatgca tcagtttcat tgcccacaca ccagaatcct actaagtttg agtattatgg 7501 cattggaaaa gctgttttct tctatcattt gttctgcttg taatttactg tgttctttca 7561 gtttttgttt tcggacatca aaatgcaaat ggatggataa gagttaataa atgatatggt 7621 ccttttgttc attctcaaat tattattatc tgttgttttt actttaatgg gttgaattta 7681 agtaagaaag gaactaacag tgtgatatta aggtgcaatg ttagacatat aaaacagtct 7741 ttcacctctc tttggttatg tcttgaattg gtttgtttct tcacttatct gtgtaatcaa 7801 gtttactatg agtctatgat caagtaatta tgcaatcaag ttaagtacag tataggcttg 7861 agctccctag gcccctaggt aattcttcgg acccaagaat gctaagccaa gaggagctgt 7921 tatcgccgtc ctcctgcttg tttctctctt tttgttgctg tttcttcatt agcgtggaca 7981 aagttttcaa ccggcctatc tgttatcatt ttcttctatt caaagactgt aatacctatt 8041 gctacctgtg gttctcactt gtgattttgg acacatatgt tcggtttatt caaatttaat 8101 cagatgcctg atgagggtac cagaaaaaat acgtgttctg gttgtttttg agttgcgatt 8161 attctatgaa atgaataaca tcgaagttat catcccagta ttttcgcatg aatgttcttt 8221 tcttctgtct tgtgcatcag tgatctagtg catgggagtt tgtattgtga tgttcgacat 8281 cacgtaactt ccactttgcc tttgctgttc gatattttaa tgacatgtca cacacacttc 8341 tgatactttt ctttcttggc tattgtgcca gcatgatgca agatgcatca cagcatcaga 8401 tatattctca tcgtcaggct ttagcagcac acgagcacgc tttgccgctt aaaagttgta 8461 cggcgcagct tagacatccc ctgtagaagt gataatcttt tcacttttcc ttaaacaaat 8521 tgagagggga aatggaacca tgtggatcag agaagctttt gtttctttac acaagaatat 8581 ttggtacagt gggggtccta tgttcgtggg ttcgtggctt ggctgcctgt cttcaaccaa 8641 gtgttttcag ttcaacatgt tagcgtgtag aaagagcaca attctgttta tctccaaggt 8701 aaaatgtggc attctgttaa agaacatgat cctgccaatt ttttaagttt caatggaaga 8761 ggaatgtaaa gctttctatg gtttgtgtac acaacacagt ggaagaggag tgcaagcttt 8821 ctatggtttg tgtgcgcgtt gtgtgtcagc acttcaattt tgttagaaaa tgaaagaaaa 8881 aaaaggatga tcatgcttat agtaaatcac tctttttcct cgccttctgt acgttttgac 8941 ttgacaagat tttaaaatct gtacatgacc tttgttttaa aattacttta tgtatttcca 9001 tctttcaagt tatgcagatg tcatcacaaa ttgttacacc aatcaccagg ctggctgttt 9061 atatattatc agaccaggct atatagagta tactatacta actgttcata ttatctggaa 9121 atcttgcttg ctacttgagc ggtaaaaggg tatagatatg agggtcccca gattagcctt 9181 ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc agcaggtctc 9241 atcaagacga tctacccgag caataatctc caggaaatca aataccttcc caagaaggtt 9301 aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa agatatattt 9361 ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca caaaccaagg 9421 caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa ggccatggag 9481 tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga acagttcata 9541 cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt ggagcacgac 9601 acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag ggcaattgag 9661 acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc agctatctgt 9721 cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat 9781 aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga tggaccccca 9841 cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat 9901 tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac 9961 ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac tctagaatga 10021 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 10081 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 10141 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 10201 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 10261 agtttagcga gagcctgacc tattgcatct cccgccgttc acagggtgtc acgttgcaag 10321 acctgcctga aaccgaactg cccgctgttc tacaaccggt cgcggaggct atggatgcga 10381 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 10441 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 10501 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 10561 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 10621 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 10681 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 10741 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccacgac 10801 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 10861 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 10921 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 10981 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaagaaat 11041 aggagctctg ggctaatcta aaacgattta tctgtggctt caagtgtatc gatcacttat 11101 gtgaggtgta attactggtg tttttggtgt gctctggttc ctttcaagtg tgttgttgcc 11161 gctcgaacta ctccgctatg taaaacggta aaacctgttg tctcattatg aaagtgaact 11221 atattatgtt ctactactac tctacttagt caattttctt caccttgatt agtgtaaata 11281 tgaatcttat attcttatgt cttaagaaat tagcacatgt gaagcctcca agtgcatatt 11341 ttctcgatcg cgagacgcac aatgcgtgag aaattcagct ggttatactc aaatatatta 11401 atatatctag cagcagctca tggagattca ggaaacttgg catccctaat ccctaccatt 11461 tccattcttc cgagattgac agttcaatac aagtacagta atctcctggt aagtttctta 11521 ttaacttgac atgtagtagt aataatttgt acgtagcata gatacataga cacaaaaatg 11581 tcctccccat tgagctagcc gattggagcc gaacaccgca ggaatgaatt tacataatct 11641 gcaaagaata aatggaatgt gcctccacag gaaaaccagc ggcagtgtgg cgttttcaag 11701 agcagccgta agtcgaagcc tattctgaat cgtagaaatc actggggcat ggtgtaatta 11761 catccgactc caacatctgt accaccctgt gcattgtagg ccgctcctct ggcaaagaac 11821 ttacacattg tttagcaaga gaaagtagag catccaaggt ctcaatctgc actccctcac 11881 aatatggatc gacaatttcc ctctcccgat tctcaccgac caggaaattc aactgccaca 11941 gagcaagtag attatttcaa gaatacatta aatcaattga aggcatacgt aattcatatc 12001 agaaaactgt ggatatgaaa tggaatgaca taaaggtcat acatacccat ccaacaatgt 12061 tcaatccctt ttcaataaat gatgcatcag taggtcgttt tccgcttagt atttcaagta 12121 gcaaaactcc aaaactgtag acgtcagtct tttcggtggc tctgccactt tgcatatact 12181 cctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt 12241 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc 12301 tttcttgtac aaagtggtga tatcccgcgg aaatacgtag aattcgggaa gcttggcact 12361 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 12421 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 12481 ttcccaacag ttgcgcagcc tgaatggcga atgctagagc aattcggcgt taattcagta 12541 cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca 12601 atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca caaaatcacc 12661 actcgataca ggcagcccat cagtccggga cggcgtcagc gggagagccg ttgtaaggcg 12721 gcagactttg ctcatgttac cgatgctatt cggaagaacg gcaactaagc tgccgggttt 12781 gaaacacgga tgatctcgcg gagggtagca tgttgattgt aacgatgaca gagcgttgct 12841 gcctgtgatc aattcgggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt 12901 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg 12961 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta 13021 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga 13081 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac atcatggggg 13141 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc 13201 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga 13261 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc 13321 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc 13381 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag 13441 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg 13501 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg 13561 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat 13621 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg 13681 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg 13741 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg 13801 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg 13861 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca 13921 aataatgtct agctagaaat tcgttcaagc cgacgccgct tcgcggcgcg gcttaactca 13981 agcgttagat gcactaagca cataattgct cacagccaaa ctatcaggtc aagtctgctt 14041 ttattatttt taagcgtgca taataagccc tacacaaatt gggagatata tcatgcatga 14101 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 14161 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 14221 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 14281 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 14341 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 14401 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 14461 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 14521 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 14581 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 14641 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 14701 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 14761 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 14821 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 14881 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 14941 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 15001 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc 15061 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg 15121 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 15181 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagggtg ccttgatgtg 15241 ggcgccggcg gtcgagtggc gacggcgcgg cttgtccgcg ccctggtaga ttgcctggcc 15301 gtaggccagc catttttgag cggccagcgg ccgcgatagg ccgacgcgaa gcggcggggc 15361 gtagggagcg cagcgaccga agggtaggcg ctttttgcag ctcttcggct gtgcgctggc 15421 cagacagtta tgcacaggcc aggcgggttt taagagtttt aataagtttt aaagagtttt 15481 aggcggaaaa atcgcctttt ttctctttta tatcagtcac ttacatgtgt gaccggttcc 15541 caatgtacgg ctttgggttc ccaatgtacg ggttccggtt cccaatgtac ggctttgggt 15601 tcccaatgta cgtgctatcc acaggaaaga gaccttttcg acctttttcc cctgctaggg 15661 caatttgccc tagcatctgc tccgtacatt aggaaccggc ggatgcttcg ccctcgatca 15721 ggttgcggta gcgcatgact aggatcgggc cagcctgccc cgcctcctcc ttcaaatcgt 15781 actccggcag gtcatttgac ccgatcagct tgcgcacggt gaaacagaac ttcttgaact 15841 ctccggcgct gccactgcgt tcgtagatcg tcttgaacaa ccatctggct tctgccttgc 15901 ctgcggcgcg gcgtgccagg cggtagagaa aacggccgat gccgggatcg atcaaaaagt 15961 aatcggggtg aaccgtcagc acgtccgggt tcttgccttc tgtgatctcg cggtacatcc 16021 aatcagctag ctcgatctcg atgtactccg gccgcccggt ttcgctcttt acgatcttgt 16081 agcggctaat caaggcttca ccctcggata ccgtcaccag gcggccgttc ttggccttct 16141 tcgtacgctg catggcaacg tgcgtggtgt ttaaccgaat gcaggtttct accaggtcgt 16201 ctttctgctt tccgccatcg gctcgccggc agaacttgag tacgtccgca acgtgtggac 16261 ggaacacgcg gccgggcttg tctcccttcc cttcccggta tcggttcatg gattcggtta 16321 gatgggaaac cgccatcagt accaggtcgt aatcccacac actggccatg ccggccggcc 16381 ctgcggaaac ctctacgtgc ccgtctggaa gctcgtagcg gatcacctcg ccagctcgtc 16441 ggtcacgctt cgacagacgg aaaacggcca cgtccatgat gctgcgacta tcgcgggtgc 16501 ccacgtcata gagcatcgga acgaaaaaat ctggttgctc gtcgcccttg ggcggcttcc 16561 taatcgacgg cgcaccggct gccggcggtt gccgggattc tttgcggatt cgatcagcgg 16621 ccgcttgcca cgattcaccg gggcgtgctt ctgcctcgat gcgttgccgc tgggcggcct 16681 gcgcggcctt caacttctcc accaggtcat cacccagcgc cgcgccgatt tgtaccgggc 16741 cggatggttt gcgaccgctc acgccgattc ctcgggcttg ggggttccag tgccattgca 16801 gggccggcag acaacccagc cgcttacgcc tggccaaccg cccgttcctc cacacatggg 16861 gcattccacg gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc ctttagccgc 16921 taaaattcat ctactcattt attcatttgc tcatttactc tggtagctgc gcgatgtatt 16981 cagatagcag ctcggtaatg gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt 17041 gatcctccgc cggcaactga aagttgaccc gcttcatggc tggcgtgtct gccaggctgg 17101 ccaacgttgc agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc 17161 ttttgctcat tttctcttta cctcattaac tcaaatgagt tttgatttaa tttcagcggc 17221 cagcgcctgg acctcgcggg cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc 17281 ggcggcggca gtgcctgggt agctcacgcg ctgcgtgata cgggactcaa gaatgggcag 17341 ctcgtacccg gccagcgcct cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg 17401 cgacacgaca aaggccgctt gtagccttcc atccgtgacc tcaatgcgct gcttaaccag 17461 ctccaccagg tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg gaatcagcac 17521 gaagtcggct gccttgatcg cggacacagc caagtccgcc gcctggggcg ctccgtcgat 17581 cactacgaag tcgcgccggc cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat 17641 gccgacaacg gttagcggtt gatcttcccg cacggccgcc caatcgcggg cactgccctg 17701 gggatcggaa tcgactaaca gaacatcggc cccggcgagt tgcagggcgc gggctagatg 17761 ggttgcgatg gtcgtcttgc ctgacccgcc tttctggtta agtacagcga taaccttcat 17821 gcgttcccct tgcgtatttg tttatttact catcgcatca tatacgcagc gaccgcatga 17881 cgcaagctgt tttactcaaa tacacatcac ctttttagac ggcggcgctc ggtttcttca 17941 gcggccaagc tggccggcca ggccgccagc ttggcatcag acaaaccggc caggatttca 18001 tgcagccgca cggttgagac gtgcgcgggc ggctcgaaca cgtacccggc cgcgatcatc 18061 tccgcctcga tctcttcggt aatgaaaaac ggttcgtcct ggccgtcctg gtgcggtttc 18121 atgcttgttc ctcttggcgt tcattctcgg cggccgccag ggcgtcggcc tcggtcaatg 18181 cgtcctcacg gaaggcaccg cgccgcctgg cctcggtggg cgtcacttcc tcgctgcgct 18241 caagtgcgcg gtacagggtc gagcgatgca cgccaagcag tgcagccgcc tctttcacgg 18301 tgcggccttc ctggtcgatc agctcgcggg cgtgcgcgat ctgtgccggg gtgagggtag 18361 ggcgggggcc aaacttcacg cctcgggcct tggcggcctc gcgcccgctc cgggtgcggt 18421 cgatgattag ggaacgctcg aactcggcaa tgccggcgaa cacggtcaac accatgcggc 18481 cggccggcgt ggtggtgtcg gcccacggct ctgccaggct acgcaggccc gcgccggcct 18541 cctggatgcg ctcggcaatg tccagtaggt cgcgggtgct gcgggccagg cggtctagcc 18601 tggtcactgt cacaacgtcg ccagggcgta ggtggtcaag catcctggcc agctccgggc 18661 ggtcgcgcct ggtgccggtg atcttctcgg aaaacagctt ggtgcagccg gccgcgtgca 18721 gttcggcccg ttggttggtc aagtcctggt cgtcggtgct gacgcgggca tagcccagca 18781 ggccagcggc ggcgctcttg ttcatggcgt aatgtctccg gttctagtcg caagtattct 18841 actttatgcg actaaaacac gcgacaagaa aacgccagga aaagggcagg gcggcagcct 18901 gtcgcgtaac ttaggacttg tgcgacatgt cgttttcaga agacggctgc actgaacgtc 18961 agaagccgac tgcactatag cagcggaggg gttggatcaa agtacttt //