LOCUS Exported 16987 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR/Cas9-mediated base editing in dicots. Contains Cas9(D10A) under CaMV 35S promoter and empty gRNA scaffold, Basta resistance. ACCESSION . VERSION . KEYWORDS pBSE901 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16987) AUTHORS Chen Y, Wang Z, Ni H, Xu Y, Chen Q, Jiang L TITLE CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis. JOURNAL Sci China Life Sci. 2017 Mar 16. doi: 10.1007/s11427-017-9021-5. PUBMED 28303459 REFERENCE 2 (bases 1 to 16987) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16987 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..25 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" primer_bind 213..235 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 227..244 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 228..244 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 957..1748 /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" misc_RNA 1900..1975 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2589..2933 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" CDS 3083..3769 /codon_start=1 /product="cytidine deaminase (C to U editing enzyme) from rat" /label=APOBEC-1 /note="can use ssDNA as a substrate (Komor et al., 2016)" /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI LWATGLK" CDS 3818..7918 /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" RBS 5601..5609 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 7931..8179 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 8192..8212 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(8869..8885) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8869..8885) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8882..8904) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8893..8909 /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)." promoter complement(8917..8947) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8962..8983 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9174..9853 /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" CDS 9898..10449 /codon_start=1 /gene="Streptomyces hygroscopicus bar" /product="phosphinothricin acetyltransferase" /label=BlpR /note="confers resistance to bialophos or phosphinothricin" /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF WQLDFSLPVPPRPVLPVTEI" polyA_signal 10456..10630 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" misc_feature 10708..10732 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 11157..11951 /codon_start=1 /gene="aphA-3" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF" rep_origin 12038..12626 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 12527..12546 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 12780..12797 /label=L4440 /note="L4440 vector, forward primer" misc_feature 12812..12952 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(12878..12897) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 13038..13060 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 13296..13490 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(13556..14629) /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(15058..15687) /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 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 61 aatctgatcc aagctcaagc tgctctagca ttcgccattc aggctgcgca actgttggga 121 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 181 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 241 cagtgccaag cttcgacttg ccttccgcac aatacatcat ttcttcttag ctttttttct 301 tcttcttcgt tcatacagtt tttttttgtt tatcagctta cattttcttg aaccgtagct 361 ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc atagtttgtc 421 ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa aacatcttca 481 ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga agagaagcag 541 gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta agttgaaaac 601 aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta tatacagcta 661 gagtcgaagt agtgattggg agaccaaccc agtggacata agcctgttcg gttcgtaagc 721 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg 781 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt 841 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat 901 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatgg 961 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc 1021 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc 1081 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa 1141 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga 1201 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc 1261 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct 1321 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata 1381 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc 1441 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg 1501 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa 1561 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc 1621 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc 1681 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg 1741 gcaaataatg tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac 1801 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg 1861 cttttattat ttttaagcgt gcataataag ccggtctcgg ttttagagct agaaatagca 1921 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 1981 tttgcaaaat tttccagatc gatttcttct tcctctgttc ttcggcgttc aatttctggg 2041 gttttctctt cgttttctgt aactgaaacc taaaatttga cctaaaaaaa atctcaaata 2101 atatgattca gtggttttgt acttttcagt tagttgagtt ttgcagttcc gatgagataa 2161 accaataagc ttgcatgcct gcaggtcaac atggtggagc acgacacact tgtctactcc 2221 aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg 2281 gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag 2341 atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc 2401 gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc 2461 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgataacatg 2521 gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa 2581 agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc 2641 ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 2701 catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 2761 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 2821 aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat 2881 ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gacctcgacc 2941 tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aagcattcta cttctattgc 3001 agcaatttaa atcatttctt ttaaagcaaa agcaattttc tgaaaatttt caccatttac 3061 gaacgatact cgagtaatct agatgtcttc cgagacagga ccggttgccg tcgaccctac 3121 tcttagaagg cgcattgagc cacacgagtt cgaggtgttc ttcgatccga gagagctgag 3181 gaaggagact tgcctccttt acgagatcaa ttggggcgga aggcactcta tttggcgcca 3241 tacctcccag aacacaaata agcatgtgga ggttaatttc atcgagaagt tcactaccga 3301 gaggtacttc tgcccaaaca cacgctgcag catcacttgg ttccttagct ggtcaccgtg 3361 cggagagtgc tctcgcgcca ttacagagtt cctgtccaga tacccgcacg ttactctttt 3421 catctacatt gccagactgt accaccatgc ggatcctcgc aacagacagg gtcttaggga 3481 cctgatcagc tcaggcgtca ccatccagat tatgacagag caggagtctg gatactgctg 3541 gcgcaacttc gtgaattact ctccttccaa tgaggctcac tggccaagat acccgcatct 3601 gtgggtcagg ctctacgtgc tcgagcttta ctgcatcatt cttggtctgc ctccatgcct 3661 caacatcctt agaaggaagc agccacagct cacattcttc actattgccc ttcagtcttg 3721 ccactaccag aggcttccgc ctcatattct gtgggcgact ggcctcaaga gcggctcaga 3781 gactccggga acatctgagt ccgctactcc tgagtctgac aagaagtact ccatcggact 3841 cgccattggt actaactccg ttggatgggc ggtcatcacc gatgagtaca aggtgcctag 3901 caagaagttc aaggttcttg gtaacacaga cagacactca atcaagaaga atctgattgg 3961 tgctctgctc ttcgattctg gagagactgc cgaggctacc aggctcaaga gaaccgcccg 4021 cagaaggtac acacgcagaa agaataggat ctgctacctt caggagattt tctctaacga 4081 gatggctaag gttgatgaca gcttcttcca tcgccttgag gagtcattcc tggtcgagga 4141 ggacaagaag cacgagagac atcctatctt cggtaacatt gtcgatgagg tggcctacca 4201 cgagaagtac ccaactatct accatcttag gaagaagctg gtggatagca ccgacaaggc 4261 ggatctccgc cttatctacc tggctctcgc ccacatgatt aagttcagag gccatttcct 4321 catcgagggc gatctcaacc cagataattc agacgtcgat aagctcttca tccagcttgt 4381 gcagacatac aatcagcttt tcgaggagaa cccgattaat gcgagcggtg ttgatgcgaa 4441 ggctatcctg tcagctagac tcagcaagtc aaggcgcctg gagaacctca tcgcccagct 4501 gccaggcgag aagaagaacg gtcttttcgg caatctgatt gcgctttctc tgggactcac 4561 cccgaacttc aagtccaatt tcgacctggc tgaggatgcc aagctccagc tgtctaagga 4621 tacatacgat gacgatctcg acaaccttct ggctcagatc ggcgaccagt acgccgatct 4681 cttccttgct gccaagaatc ttagcgatgc catcctcctt tcagacattc tgagagttaa 4741 cactgagatt accaaggctc cgctgtctgc ctccatgatc aagagatacg atgagcacca 4801 tcaggacctc actctgctca aggcgctggt ccgccagcag ctccctgaga agtacaagga 4861 gatcttcttc gaccagtcta agaacggcta cgcgggttac attgatggtg gcgctagcca 4921 ggaggagttc tacaagttca tcaagccaat tctggagaag atggatggca ctgaggagct 4981 tctggtcaag ctcaataggg aggatctcct taggaagcag cgcaccttcg acaacggatc 5041 tatccctcac cagattcatc ttggagagct gcacgccatc ctcagaaggc aggaggattt 5101 ctacccattc cttaaggaca accgcgagaa gatcgagaag attctgactt tcagaatccc 5161 ttactacgtt ggcccgctcg ctagaggcaa ctctaggttc gcgtggatga ccaggaagtc 5221 agaggagact atcacccctt ggaacttcga ggaggtggtt gacaagggag ccagcgcgca 5281 gtcattcatt gagcgcatga ctaatttcga taagaacctg cctaatgaga aggtcctccc 5341 aaagcatagc ctgctctacg agtacttcac tgtgtacaac gagcttacca aggtgaagta 5401 tgtgacagag ggcatgcgca agccggcttt cctttcagga gagcagaaga aggccatcgt 5461 ggatcttctg ttcaagacta atagaaaggt caccgtgaag cagctgaagg aggattactt 5521 caagaagatt gagtgcttcg actctgttga gatctccggt gtcgaggata ggttcaacgc 5581 ttccctcggc acctaccacg acctccttaa gatcattaag gacaaggatt tcctggataa 5641 cgaggagaat gaggacatcc tcgaggatat tgtgctgaca ctcactcttt tcgaggacag 5701 ggagatgatc gaggagcgcc ttaagacata cgcgcatctg ttcgacgata aggttatgaa 5761 gcagctcaag cgcagaaggt acactggatg gggtagactc tctaggaagc tcatcaacgg 5821 catcagagat aagcagtctg gcaagactat tctcgatttc cttaagtccg acggcttcgc 5881 taacaggaat ttcatgcagc tcattcacga cgattctctt actttcaagg aggacatcca 5941 gaaggcgcag gttagcggcc agggagattc actgcacgag catatcgcga acctcgctgg 6001 ctcccctgct atcaagaagg gcatcctcca gaccgttaag gtcgtggatg agctggttaa 6061 ggtcatgggc agacataagc cagagaacat cgtcattgag atggccaggg agaatcagac 6121 aactcagaag ggacagaaga actctaggga gcgcatgaag agaatcgagg agggtattaa 6181 ggagcttggc tcccagatcc tgaaggagca cccggtggag aacacacagc tgcagaatga 6241 gaagctgtac ctctactacc tccagaatgg ccgcgacatg tatgtggatc aggagcttga 6301 cattaacaga ctttctgact acgatgtgga ccatatcgtt ccacagtctt tccttaagga 6361 cgattccatt gataataagg tgctgactag atccgataag aacaggggaa agtctgacaa 6421 tgttccgtcc gaggaggttg tcaagaagat gaagaactac tggaggcagc tgctcaatgc 6481 taagctcatc acccagagga agttcgacaa ccttacaaag gccgagcgcg gaggtctgag 6541 cgagcttgat aaggcgggtt tcattaagag acagctcgtt gagacaaggc agatcactaa 6601 gcacgtcgcc cagattcttg actcaaggat gaacaccaag tacgacgaga atgataagct 6661 gatccgcgag gtgaaggtta ttacactgaa gagcaagctc gtttcagatt tcagaaagga 6721 cttccagttc tacaaggtca gggagatcaa caattaccac catgcccatg atgcgtacct 6781 caacgcggtg gttggtactg ctcttattaa gaagtacccg aagctggagt ctgagttcgt 6841 gtacggcgat tacaaggtgt acgacgttag aaagatgatc gctaagagcg agcaggagat 6901 tggcaaggct accgccaagt acttcttcta ctcaaacatt atgaatttct tcaagacaga 6961 gatcactctc gcgaacggcg agatcagaaa gaggccactt attgagacta acggcgagac 7021 aggagagatc gtctgggata agggtcgcga cttcgctact gtcagaaagg tgctctctat 7081 gccgcaggtt aatattgtca agaagactga ggtgcagacc ggcggattct ctaaggagtc 7141 cattctccct aagaggaact ccgacaagct catcgcccgc aagaaggatt gggaccctaa 7201 gaagtacggt ggcttcgata gcccaaccgt cgcttactca gtgcttgtcg tggccaaggt 7261 cgagaaggga aagagcaaga agctgaagtc agtgaaggag cttctgggta tcacaattat 7321 ggagaggtct tccttcgaga agaatcctat cgacttcctc gaggcgaagg gctacaagga 7381 ggttaagaag gatcttatca ttaagctgcc aaagtactca cttttcgagc tggagaacgg 7441 acgcaagaga atgctggcgt ctgctggaga gcttcagaag ggtaatgagc ttgctctgcc 7501 gtctaagtat gtgaacttcc tctaccttgc ctctcattac gagaagctca agggctcccc 7561 tgaggacaac gagcagaagc agctgttcgt cgagcagcac aagcattacc tcgatgagat 7621 cattgagcag attagcgagt tctcaaagag agtgatcctc gccgatgcga atctcgacaa 7681 ggttcttagc gcgtacaaca agcaccgcga taagccaatc agagagcagg ctgagaatat 7741 cattcatctc ttcaccctta caaacctggg tgctccggcg gctttcaagt acttcgatac 7801 cacaattgac aggaagcgct acacttcaac caaggaggtg ctggacgcca ccctcatcca 7861 ccagtctatt actggcctct acgagactag gatcgatctc tcccagcttg gtggtgactc 7921 tggcggatcc accaacctca gcgatatcat tgagaaggag acaggcaagc agcttgttat 7981 ccaggagtca attctgatgc tcccggagga ggtggaggag gttattggca ataagcctga 8041 gtctgatatc ctcgtgcata ctgcctacga tgagagcacc gacgagaacg ttatgctcct 8101 tacatcagac gcgcctgagt acaagccttg ggctctcgtc attcaggatt ccaacggaga 8161 gaataagatc aagatgctta gcggtggctc tcctaagaag aagagaaagg tgtgagctca 8221 gagctttcgt tcgtatcatc ggtttcgaca acgttcgtca agttcaatgc atcagtttca 8281 ttgcgcacac accagaatcc tactgagttt gagtattatg gcattgggaa aactgttttt 8341 cttgtaccat ttgttgtgct tgtaatttac tgtgtttttt attcggtttt cgctatcgaa 8401 ctgtgaaatg gaaatggatg gagaagagtt aatgaatgat atggtccttt tgttcattct 8461 caaattaata ttatttgttt tttctcttat ttgttgtgtg ttgaatttga aattataaga 8521 gatatgcaaa cattttgttt tgagtaaaaa tgtgtcaaat cgtggcctct aatgaccgaa 8581 gttaatatga ggagtaaaac acttgtagtt gtaccattat gcttattcac taggcaacaa 8641 atatattttc agacctagaa aagctgcaaa tgttactgaa tacaagtatg tcctcttgtg 8701 ttttagacat ttatgaactt tcctttatgt aattttccag aatccttgtc agattctaat 8761 cattgcttta taattatagt tatactcatg gatttgtagt tgagtatgaa aatatttttt 8821 aatgcatttt atgacttgcc aattgattga caacgaattc gtaatcatgt catagctgtt 8881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 8941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 9001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 9061 ggggagaggc ggtttgcgta ttggctagag cagcttgcca acatggtgga gcacgacact 9121 ctcgtctact ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact 9181 tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac 9241 ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa 9301 ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc 9361 acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcmaagca agtggattga 9421 tgtgacagac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt 9481 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct 9541 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg 9601 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga 9661 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 9721 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 9781 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 9841 gaggacacgc tgaaatcacc agtctctctc tacaaatcta tctctctcga gtctaccatg 9901 agcccagaac gacgcccggc cgacatccgc cgtgccaccg aggcggacat gccggcggtc 9961 tgcaccatcg tcaaccacta catcgagaca agcacggtca acttccgtac cgagccgcag 10021 gaaccgcagg agtggacgga cgacctcgtc cgtctgcggg agcgctatcc ctggctcgtc 10081 gccgaggtgg acggcgaggt cgccggcatc gcctacgcgg gcccctggaa ggcacgcaac 10141 gcctacgact ggacggccga gtcgaccgtg tacgtctccc cccgccacca gcggacggga 10201 ctgggctcca cgctctacac ccacctgctg aagtccctgg aggcacaggg cttcaagagc 10261 gtggtcgctg tcatcgggct gcccaacgac ccgagcgtgc gcatgcacga ggcgctcgga 10321 tatgcccccc gcggcatgct gcgggcggcc ggcttcaagc acgggaactg gcatgacgtg 10381 ggtttctggc agctggactt cagcctgccg gtaccgcccc gtccggtcct gcccgtcacc 10441 gagatttgac tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag 10501 ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt 10561 atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa 10621 aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt 10681 gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag 10741 ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc 10801 agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc 10861 gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg 10921 agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca ccgcggtttc 10981 aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt tgaaaaagct 11041 gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt cgtcttgtta 11101 taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa taataaatgg 11161 ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag 11221 atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat 11281 atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg 11341 acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc 11401 atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt 11461 atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct 11521 ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg 11581 aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag 11641 acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag 11701 aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca 11761 aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg 11821 ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc gagctatttt 11881 ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg 11941 aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt tcgttccact 12001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 12061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 12121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 12181 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 12241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 12301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 12361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 12421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 12481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 12541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 12601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 12661 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 12721 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 12781 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 12841 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 12901 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 12961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 13021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 13081 acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt 13141 gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg ccagcggccg 13201 cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt 13261 tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg cgggttttaa 13321 gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc tcttttatat 13381 cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca atgtacgggt 13441 tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca ggaaacagac 13501 cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc gtacattagg 13561 aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg atcgggccag 13621 cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg atcagcttgc 13681 gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg tagatcgtct 13741 tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac 13801 ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg tccgggttct 13861 tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg tactccggcc 13921 gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc tcggataccg 13981 tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta 14041 accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct cgccggcaga 14101 acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct cccttccctt 14161 cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc aggtcgtaat 14221 cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg tctggaagct 14281 cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa acggccacgt 14341 ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg aaaaaatctg 14401 gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc 14461 gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg cgtgcttctg 14521 cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc aggtcatcac 14581 ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgctcacg ccgattcctc 14641 gggcttgggg gttccagtgc cattgcaggg ccggcaggca acccagccgc ttacgcctgg 14701 ccaaccgccc gttcctccac acatggggca ttccacggcg tcggtgcctg gttgttcttg 14761 attttccatg ccgcctcctt tagccgctaa aattcatcta ctcatttatt catttgctca 14821 tttactctgg tagctgcgcg atgtattcag atagcagctc ggtaatggtc ttgccttggc 14881 gtaccgcgta catcttcagc ttggtgtgat cctccgccgg caactgaaag ttgacccgct 14941 tcatggctgg cgtgtctgcc aggctggcca acgttgcagc cttgctgctg cgtgcgctcg 15001 gacggccggc acttagcgtg tttgtgcttt tgctcatttt ctctttacct cattaactca 15061 aatgagtttt gatttaattt cagcggccag cgcctggacc tcgcgggcag cgtcgccctc 15121 gggttctgat tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc tcacgcgctg 15181 cgtgatacgg gactcaagaa tgggcagctc gtacccggcc agcgcctcgg caacctcacc 15241 gccgatgcgc gtgcctttga tcgcccgcga cacgacaaag gccgcttgta gccttccatc 15301 cgtgacctca atgcgctgct taaccagctc caccaggtcg gcggtggccc atatgtcgta 15361 agggcttggc tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg acacagccaa 15421 gtccgccgcc tggggcgctc cgtcgatcac tacgaagtcg cgccggccga tggccttcac 15481 gtcgcggtca atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat cttcccgcac 15541 ggccgcccaa tcgcgggcac tgccctgggg atcggaatcg actaacagaa catcggcccc 15601 ggcgagttgc agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg acccgccttt 15661 ctggttaagt acagcgataa ccttcatgcg ttccccttgc gtatttgttt atttactcat 15721 cgcatcatat acgcagcgac cgcatgacgc aagctgtttt actcaaatac acatcacctt 15781 tttagacggc ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc cgccagcttg 15841 gcatcagaca aaccggccag gatttcatgc agccgcacgg ttgagacgtg cgcgggcggc 15901 tcgaacacgt acccggccgc gatcatctcc gcctcgatct cttcggtaat gaaaaacggt 15961 tcgtcctggc cgtcctggtg cggtttcatg cttgttcctc ttggcgttca ttctcggcgg 16021 ccgccagggc gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc cgcctggcct 16081 cggtgggcgt cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag cgatgcacgc 16141 caagcagtgc agccgcctct ttcacggtgc ggccttcctg gtcgatcagc tcgcgggcgt 16201 gcgcgatctg tgccggggtg agggtagggc gggggccaaa cttcacgcct cgggccttgg 16261 cggcctcgcg cccgctccgg gtgcggtcga tgattaggga acgctcgaac tcggcaatgc 16321 cggcgaacac ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc cacggctctg 16381 ccaggctacg caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc agtaggtcgc 16441 gggtgctgcg ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca gggcgtaggt 16501 ggtcaagcat cctggccagc tccgggcggt cgcgcctggt gccggtgatc ttctcggaaa 16561 acagcttggt gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag tcctggtcgt 16621 cggtgctgac gcgggcatag cccagcaggc cagcggcggc gctcttgttc atggcgtaat 16681 gtctccggtt ctagtcgcaa gtattctact ttatgcgact aaaacacgcg acaagaaaac 16741 gccaggaaaa gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc gacatgtcgt 16801 tttcagaaga cggctgcact gaacgtcaga agccgactgc actatagcag cggaggggtt 16861 ggatcaaagt actttgatcc cgaggggaac cctgtggttg gcatgcacat acaaatggac 16921 gaacggataa accttttcac gcccttttaa atatccgatt attctaataa acgctctttt 16981 ctcttag //