LOCUS Exported 17949 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses CRISPR base editor in maize. ACCESSION . VERSION . KEYWORDS pB-nCas9-PBE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 17949) AUTHORS Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C TITLE Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. JOURNAL Nat Biotechnol. 2017 May;35(5):438-440. doi: 10.1038/nbt.3811. Epub 2017 Feb 27. PUBMED 28244994 REFERENCE 2 (bases 1 to 17949) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..17949 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 21..45 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" primer_bind 233..255 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 247..264 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 248..264 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 331..710 /label=OsU3 promoter /note="Oryza sativa (rice) snRNA U3 promoter" CDS 990..1781 /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 1933..2008 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2312..4303 /label=Ubi promoter /note="maize polyubiquitin gene promoter" CDS 4314..4334 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4335..5018 /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="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL WATGLK" CDS 5070..9170 /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" CDS 9171..9218 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 9240..9488 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 9501..9521 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(10179..10195) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10179..10195) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(10192..10214) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 10203..10219 /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(10227..10257) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 10272..10293 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 10484..10829 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" primer_bind 10772..10792 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 10880..11431 /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 11438..11612 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" misc_feature 11690..11714 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 12139..12933 /codon_start=1 /gene="aphA-3" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF" rep_origin 13020..13608 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 13509..13528 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 13762..13779 /label=L4440 /note="L4440 vector, forward primer" misc_feature 13794..13934 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(13860..13879) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 14020..14042 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 14278..14472 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(14538..15611) /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(16040..16669) /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 taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 61 aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 121 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 181 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 241 cgacgttgta aaacgacggc cagtgccaag cttagtaatt catccaggtc tccaagttct 301 aggattttca gaactgcaac ttattttatc aaggaatctt taaacatacg aacagatcac 361 ttaaagttct tctgaagcaa cttaaagtta tcaggcatgc atggatcttg gaggaatcag 421 atgtgcagtc agggaccata gcacaagaca ggcgtcttct actggtgcta ccagcaaatg 481 ctggaagccg ggaacactgg gtacgttgga aaccacgtga tgtgaagaag taagataaac 541 tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt attgcagtat 601 gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc acctcggcta 661 tccacataga tcaaagctga tttaaaagag ttgtgcagat gatccgtggc gtgagaccaa 721 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac 781 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca 841 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag 901 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg 961 gcagtcgccc taaaacaaag ttaaacatca tgggggaagc ggtgatcgcc gaagtatcga 1021 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg 1081 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc 1141 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt 1201 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg 1261 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag 1321 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc 1381 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg 1441 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa 1501 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt 1561 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact 1621 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt 1681 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc 1741 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctagct agaaattcgt 1801 tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg ttagatgcac taagcacata 1861 attgctcaca gccaaactat caggtcaagt ctgcttttat tatttttaag cgtgcataat 1921 aagccggtct cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca 1981 acttgaaaaa gtggcaccga gtcggtgctt ttttttttcg ttttgcattg agttttctcc 2041 gtcgcatgtt tgcagtttta ttttccgttt tgcattgaaa tttctccgtc tcatgtttgc 2101 agcgtgttca aaaagtacgc agctgtattt cacttattta cggcgccaca ttttcatgcc 2161 gtttgtgcca actatcccga gctagtgaat acagcttggc ttcacacaac actggtgacc 2221 cgctgacctg ctcgtacctc gtaccgtcgt acggcacagc atttggaatt aaagggtgtg 2281 atcgatactg cttgctgcta agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc 2341 ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt 2401 tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa actttactct 2461 acgaataata taatctatag tactacaata atatcagtgt tttagagaat catataaatg 2521 aacagttaga catggtctaa aggacaattg agtattttga caacaggact ctacagtttt 2581 atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac 2641 ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa 2701 tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct 2761 attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa 2821 attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag 2881 tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc 2941 agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg 3001 acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt 3061 gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc 3121 accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc 3181 gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca 3241 cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc 3301 cgctcgtcct cccccccccc cctctctacc ttctctagat cggcgttccg gtccatggtt 3361 agggcccggt agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc 3421 gtgctgctag cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac 3481 ttgccagtgt ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc 3541 gatttcatga ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc 3601 aatatatgcc gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg 3661 tgatgatgtg gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta 3721 cctggtggat ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga 3781 attgaagatg atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt 3841 actgatgcat atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg 3901 gcggtcgttc attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt 3961 attaattttg gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga 4021 tggaaatatc gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca 4081 tgatggcata tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata 4141 aacaagtatg ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca 4201 gctatatgtg gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt 4261 cttttgtcga tgctcaccct gttgtttggt gttacttctg cagccctagg atgccaaaga 4321 agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc ctgcggcgga 4381 gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc aaggagacgt 4441 gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac accagccaaa 4501 acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag aggtacttct 4561 gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc ggcgagtgct 4621 ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc atctacatcg 4681 ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac ctgatctcca 4741 gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg aggaacttcg 4801 tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc tgggtgcgcc 4861 tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc aacatcctga 4921 ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc cactaccaga 4981 ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag acgccaggca 5041 cctccgagag cgctacgcct gaacttaagg acaagaagta ctcgatcggc ctcgccatcg 5101 ggacgaactc agttggctgg gccgtgatca ccgacgagta caaggtgccc tctaagaagt 5161 tcaaggtcct ggggaacacc gaccgccatt ccatcaagaa gaacctcatc ggcgctctcc 5221 tgttcgacag cggggagacc gctgaggcta cgaggctcaa gagaaccgct aggcgccggt 5281 acacgagaag gaagaacagg atctgctacc tccaagagat tttctccaac gagatggcca 5341 aggttgacga ttcattcttc caccgcctgg aggagtcttt cctcgtggag gaggataaga 5401 agcacgagcg gcatcccatc ttcggcaaca tcgtggacga ggttgcctac cacgagaagt 5461 accctacgat ctaccatctg cggaagaagc tcgtggactc caccgataag gcggacctca 5521 gactgatcta cctcgctctg gcccacatga tcaagttccg cggccatttc ctgatcgagg 5581 gggatctcaa cccagacaac agcgatgttg acaagctgtt catccaactc gtgcagacct 5641 acaaccaact cttcgaggag aacccgatca acgcctctgg cgtggacgcg aaggctatcc 5701 tgtccgcgag gctctcgaag tccaggaggc tggagaacct gatcgctcag ctcccaggcg 5761 agaagaagaa cggcctgttc gggaacctca tcgctctcag cctggggctc accccgaact 5821 tcaagtcgaa cttcgatctc gctgaggacg ccaagctgca actctccaag gacacctacg 5881 acgatgacct cgataacctc ctggcccaga tcggcgatca atacgcggac ctgttcctcg 5941 ctgccaagaa cctgtcggac gccatcctcc tgtcagatat cctccgcgtg aacaccgaga 6001 tcacgaaggc tccactctct gcctccatga tcaagcgcta cgacgagcac catcaggatc 6061 tgaccctcct gaaggcgctg gtccgccaac agctcccgga gaagtacaag gagattttct 6121 tcgatcagtc gaagaacggc tacgctgggt acatcgacgg cggggcctca caagaggagt 6181 tctacaagtt catcaagcca atcctggaga agatggacgg cacggaggag ctcctggtga 6241 agctcaacag ggaggacctc ctgcggaagc agagaacctt cgataacggc agcatccccc 6301 accaaatcca tctcggggag ctgcacgcca tcctgagaag gcaagaggac ttctaccctt 6361 tcctcaagga taaccgggag aagatcgaga agatcctgac cttcagaatc ccatactacg 6421 tcggccctct cgcgcggggg aactcaagat tcgcttggat gacccgcaag tctgaggaga 6481 ccatcacgcc gtggaacttc gaggaggtgg tggacaaggg cgctagcgct cagtcgttca 6541 tcgagaggat gaccaacttc gacaagaacc tgcccaacga gaaggtgctc cctaagcact 6601 cgctcctgta cgagtacttc accgtctaca acgagctcac gaaggtgaag tacgtcaccg 6661 agggcatgcg caagccagcg ttcctgtccg gggagcagaa gaaggctatc gtggacctcc 6721 tgttcaagac caaccggaag gtcacggtta agcaactcaa ggaggactac ttcaagaaga 6781 tcgagtgctt cgattcggtc gagatcagcg gcgttgagga ccgcttcaac gccagcctcg 6841 ggacctacca cgatctcctg aagatcatca aggataagga cttcctggac aacgaggaga 6901 acgaggatat cctggaggac atcgtgctga ccctcacgct gttcgaggac agggagatga 6961 tcgaggagcg cctgaagacg tacgcccatc tcttcgatga caaggtcatg aagcaactca 7021 agcgccggag atacaccggc tgggggaggc tgtcccgcaa gctcatcaac ggcatccggg 7081 acaagcagtc cgggaagacc atcctcgact tcctcaagag cgatggcttc gccaacagga 7141 acttcatgca actgatccac gatgacagcc tcaccttcaa ggaggatatc caaaaggctc 7201 aagtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcccccg 7261 cgatcaagaa gggcatcctc cagaccgtga aggttgtgga cgagctcgtg aaggtcatgg 7321 gccggcacaa gcctgagaac atcgtcatcg agatggccag agagaaccaa accacgcaga 7381 aggggcaaaa gaactctagg gagcgcatga agcgcatcga ggagggcatc aaggagctgg 7441 ggtcccaaat cctcaaggag cacccagtgg agaacaccca actgcagaac gagaagctct 7501 acctgtacta cctccagaac ggcagggata tgtacgtgga ccaagagctg gatatcaacc 7561 gcctcagcga ttacgacgtc gatcatatcg ttccccagtc tttcctgaag gatgactcca 7621 tcgacaacaa ggtcctcacc aggtcggaca agaaccgcgg caagtcagat aacgttccat 7681 ctgaggaggt cgttaagaag atgaagaact actggaggca gctcctgaac gccaagctga 7741 tcacgcaaag gaagttcgac aacctcacca aggctgagag aggcgggctc tcagagctgg 7801 acaaggccgg cttcatcaag cggcagctgg tcgagaccag acaaatcacg aagcacgttg 7861 cgcaaatcct cgactctcgg atgaacacga agtacgatga gaacgacaag ctgatcaggg 7921 aggttaaggt gatcaccctg aagtctaagc tcgtctccga cttcaggaag gatttccagt 7981 tctacaaggt tcgcgagatc aacaactacc accatgccca tgacgcttac ctcaacgctg 8041 tggtcggcac cgctctgatc aagaagtacc caaagctgga gtccgagttc gtgtacgggg 8101 actacaaggt ttacgatgtg cgcaagatga tcgccaagtc ggagcaagag atcggcaagg 8161 ctaccgccaa gtacttcttc tactcaaaca tcatgaactt cttcaagacc gagatcacgc 8221 tggccaacgg cgagatccgg aagagaccgc tcatcgagac caacggcgag acgggggaga 8281 tcgtgtggga caagggcagg gatttcgcga ccgtccgcaa ggttctctcc atgccccagg 8341 tgaacatcgt caagaagacc gaggtccaaa cgggcgggtt ctcaaaggag tctatcctgc 8401 ctaagcggaa cagcgacaag ctcatcgcca gaaagaagga ctgggaccca aagaagtacg 8461 gcgggttcga cagccctacc gtggcctact cggtcctggt tgtggcgaag gttgagaagg 8521 gcaagtccaa gaagctcaag agcgtgaagg agctcctggg gatcaccatc atggagaggt 8581 ccagcttcga gaagaaccca atcgacttcc tggaggccaa gggctacaag gaggtgaaga 8641 aggacctgat catcaagctc ccgaagtact ctctcttcga gctggagaac ggcaggaaga 8701 gaatgctggc ttccgctggc gagctccaga aggggaacga gctcgcgctg ccaagcaagt 8761 acgtgaactt cctctacctg gcttcccact acgagaagct caagggcagc ccggaggaca 8821 acgagcaaaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcatcgagc 8881 aaatctccga gttcagcaag cgcgtgatcc tcgccgacgc gaacctggat aaggtcctct 8941 ccgcctacaa caagcaccgg gacaagccca tcagagagca agcggagaac atcatccatc 9001 tcttcaccct gacgaacctc ggcgctcctg ctgctttcaa gtacttcgac accacgatcg 9061 atcggaagag atacacctcc acgaaggagg tcctggacgc gaccctcatc caccagtcga 9121 tcaccggcct gtacgagacg aggatcgacc tctcacaact cggcggggat aagagacccg 9181 cagcaaccaa gaaggcaggg caagcaaaga agaagaagac gcgtgactcc ggcggcagca 9241 ccaacctgtc cgacatcatc gagaaggaga cgggcaagca actcgtgatc caggagagca 9301 tcctcatgct gccagaggag gtggaggagg tcatcggcaa caagccagag tccgacatcc 9361 tggtgcacac cgcctacgac gagtccaccg acgagaacgt catgctcctg accagcgacg 9421 ccccagagta caagccatgg gccctcgtca tccaggacag caacggggag aacaagatca 9481 agatgctgtc gggggggagc ccaaagaaga agcggaaggt gtagagctca gagctttcgt 9541 tcgtatcatc ggtttcgaca acgttcgtca agttcaatgc atcagtttca ttgcgcacac 9601 accagaatcc tactgagttt gagtattatg gcattgggaa aactgttttt cttgtaccat 9661 ttgttgtgct tgtaatttac tgtgtttttt attcggtttt cgctatcgaa ctgtgaaatg 9721 gaaatggatg gagaagagtt aatgaatgat atggtccttt tgttcattct caaattaata 9781 ttatttgttt tttctcttat ttgttgtgtg ttgaatttga aattataaga gatatgcaaa 9841 cattttgttt tgagtaaaaa tgtgtcaaat cgtggcctct aatgaccgaa gttaatatga 9901 ggagtaaaac acttgtagtt gtaccattat gcttattcac taggcaacaa atatattttc 9961 agacctagaa aagctgcaaa tgttactgaa tacaagtatg tcctcttgtg ttttagacat 10021 ttatgaactt tcctttatgt aattttccag aatccttgtc agattctaat cattgcttta 10081 taattatagt tatactcatg gatttgtagt tgagtatgaa aatatttttt aatgcatttt 10141 atgacttgcc aattgattga caacgaattc gtaatcatgt catagctgtt tcctgtgtga 10201 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 10261 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 10321 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 10381 ggtttgcgta ttggctagag cagcttgcca acatggtgga gcacgacact ctcgtctact 10441 ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa 10501 gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa 10561 ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta 10621 tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca 10681 tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct 10741 ccactgacgt aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat 10801 aaggaagttc atttcatttg gagaggacac gctgaaatca ccagtctctc tctacaaatc 10861 tatctctctc gagtctacca tgagcccaga acgacgcccg gccgacatcc gccgtgccac 10921 cgaggcggac atgccggcgg tctgcaccat cgtcaaccac tacatcgaga caagcacggt 10981 caacttccgt accgagccgc aggaaccgca ggagtggacg gacgacctcg tccgtctgcg 11041 ggagcgctat ccctggctcg tcgccgaggt ggacggcgag gtcgccggca tcgcctacgc 11101 gggcccctgg aaggcacgca acgcctacga ctggacggcc gagtcgaccg tgtacgtctc 11161 cccccgccac cagcggacgg gactgggctc cacgctctac acccacctgc tgaagtccct 11221 ggaggcacag ggcttcaaga gcgtggtcgc tgtcatcggg ctgcccaacg acccgagcgt 11281 gcgcatgcac gaggcgctcg gatatgcccc ccgcggcatg ctgcgggcgg ccggcttcaa 11341 gcacgggaac tggcatgacg tgggtttctg gcagctggac ttcagcctgc cggtaccgcc 11401 ccgtccggtc ctgcccgtca ccgagatttg actcgagttt ctccataata atgtgtgagt 11461 agttcccaga taagggaatt agggttccta tagggtttcg ctcatgtgtt gagcatataa 11521 gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc 11581 taaaaccaaa atccagtact aaaatccaga tcccccgaat taattcggcg ttaattcagt 11641 acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg tttacaccac 11701 aatatatcct gccaccagcc agccaacagc tccccgaccg gcagctcggc acaaaatcac 11761 cactcgatac aggcagccca tcagtccggg acggcgtcag cgggagagcc gttgtaaggc 11821 ggcagacttt gctcatgtta ccgatgctat tcggaagaac ggcaactaag ctgccgggtt 11881 tgaaacacgg atgatctcgc ggagggtagc atgttgattg taacgatgac agagcgttgc 11941 tgcctgtgat caccgcggtt tcaaaatcgg ctccgtcgat actatgttat acgccaactt 12001 tgaaaacaac tttgaaaaag ctgttttctg gtatttaagg ttttagaatg caaggaacag 12061 tgaattggag ttcgtcttgt tataattagc ttcttggggt atctttaaat actgtagaaa 12121 agaggaagga aataataaat ggctaaaatg agaatatcac cggaattgaa aaaactgatc 12181 gaaaaatacc gctgcgtaaa agatacggaa ggaatgtctc ctgctaaggt atataagctg 12241 gtgggagaaa atgaaaacct atatttaaaa atgacggaca gccggtataa agggaccacc 12301 tatgatgtgg aacgggaaaa ggacatgatg ctatggctgg aaggaaagct gcctgttcca 12361 aaggtcctgc actttgaacg gcatgatggc tggagcaatc tgctcatgag tgaggccgat 12421 ggcgtccttt gctcggaaga gtatgaagat gaacaaagcc ctgaaaagat tatcgagctg 12481 tatgcggagt gcatcaggct ctttcactcc atcgacatat cggattgtcc ctatacgaat 12541 agcttagaca gccgcttagc cgaattggat tacttactga ataacgatct ggccgatgtg 12601 gattgcgaaa actgggaaga agacactcca tttaaagatc cgcgcgagct gtatgatttt 12661 ttaaagacgg aaaagcccga agaggaactt gtcttttccc acggcgacct gggagacagc 12721 aacatctttg tgaaagatgg caaagtaagt ggctttattg atcttgggag aagcggcagg 12781 gcggacaagt ggtatgacat tgccttctgc gtccggtcga tcagggagga tatcggggaa 12841 gaacagtatg tcgagctatt ttttgactta ctggggatca agcctgattg ggagaaaata 12901 aaatattata ttttactgga tgaattgttt tagtacctag aatgcatgac caaaatccct 12961 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 13021 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 13081 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 13141 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 13201 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 13261 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 13321 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 13381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 13441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 13501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 13561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 13621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 13681 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 13741 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 13801 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 13861 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 13921 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 13981 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 14041 ggttttcacc gtcatcaccg aaacgcgcga ggcagggtgc cttgatgtgg gcgccggcgg 14101 tcgagtggcg acggcgcggc ttgtccgcgc cctggtagat tgcctggccg taggccagcc 14161 atttttgagc ggccagcggc cgcgataggc cgacgcgaag cggcggggcg tagggagcgc 14221 agcgaccgaa gggtaggcgc tttttgcagc tcttcggctg tgcgctggcc agacagttat 14281 gcacaggcca ggcgggtttt aagagtttta ataagtttta aagagtttta ggcggaaaaa 14341 tcgccttttt tctcttttat atcagtcact tacatgtgtg accggttccc aatgtacggc 14401 tttgggttcc caatgtacgg gttccggttc ccaatgtacg gctttgggtt cccaatgtac 14461 gtgctatcca caggaaacag accttttcga cctttttccc ctgctagggc aatttgccct 14521 agcatctgct ccgtacatta ggaaccggcg gatgcttcgc cctcgatcag gttgcggtag 14581 cgcatgacta ggatcgggcc agcctgcccc gcctcctcct tcaaatcgta ctccggcagg 14641 tcatttgacc cgatcagctt gcgcacggtg aaacagaact tcttgaactc tccggcgctg 14701 ccactgcgtt cgtagatcgt cttgaacaac catctggctt ctgccttgcc tgcggcgcgg 14761 cgtgccaggc ggtagagaaa acggccgatg ccgggatcga tcaaaaagta atcggggtga 14821 accgtcagca cgtccgggtt cttgccttct gtgatctcgc ggtacatcca atcagctagc 14881 tcgatctcga tgtactccgg ccgcccggtt tcgctcttta cgatcttgta gcggctaatc 14941 aaggcttcac cctcggatac cgtcaccagg cggccgttct tggccttctt cgtacgctgc 15001 atggcaacgt gcgtggtgtt taaccgaatg caggtttcta ccaggtcgtc tttctgcttt 15061 ccgccatcgg ctcgccggca gaacttgagt acgtccgcaa cgtgtggacg gaacacgcgg 15121 ccgggcttgt ctcccttccc ttcccggtat cggttcatgg attcggttag atgggaaacc 15181 gccatcagta ccaggtcgta atcccacaca ctggccatgc cggccggccc tgcggaaacc 15241 tctacgtgcc cgtctggaag ctcgtagcgg atcacctcgc cagctcgtcg gtcacgcttc 15301 gacagacgga aaacggccac gtccatgatg ctgcgactat cgcgggtgcc cacgtcatag 15361 agcatcggaa cgaaaaaatc tggttgctcg tcgcccttgg gcggcttcct aatcgacggc 15421 gcaccggctg ccggcggttg ccgggattct ttgcggattc gatcagcggc cgcttgccac 15481 gattcaccgg ggcgtgcttc tgcctcgatg cgttgccgct gggcggcctg cgcggccttc 15541 aacttctcca ccaggtcatc acccagcgcc gcgccgattt gtaccgggcc ggatggtttg 15601 cgaccgctca cgccgattcc tcgggcttgg gggttccagt gccattgcag ggccggcagg 15661 caacccagcc gcttacgcct ggccaaccgc ccgttcctcc acacatgggg cattccacgg 15721 cgtcggtgcc tggttgttct tgattttcca tgccgcctcc tttagccgct aaaattcatc 15781 tactcattta ttcatttgct catttactct ggtagctgcg cgatgtattc agatagcagc 15841 tcggtaatgg tcttgccttg gcgtaccgcg tacatcttca gcttggtgtg atcctccgcc 15901 ggcaactgaa agttgacccg cttcatggct ggcgtgtctg ccaggctggc caacgttgca 15961 gccttgctgc tgcgtgcgct cggacggccg gcacttagcg tgtttgtgct tttgctcatt 16021 ttctctttac ctcattaact caaatgagtt ttgatttaat ttcagcggcc agcgcctgga 16081 cctcgcgggc agcgtcgccc tcgggttctg attcaagaac ggttgtgccg gcggcggcag 16141 tgcctgggta gctcacgcgc tgcgtgatac gggactcaag aatgggcagc tcgtacccgg 16201 ccagcgcctc ggcaacctca ccgccgatgc gcgtgccttt gatcgcccgc gacacgacaa 16261 aggccgcttg tagccttcca tccgtgacct caatgcgctg cttaaccagc tccaccaggt 16321 cggcggtggc ccatatgtcg taagggcttg gctgcaccgg aatcagcacg aagtcggctg 16381 ccttgatcgc ggacacagcc aagtccgccg cctggggcgc tccgtcgatc actacgaagt 16441 cgcgccggcc gatggccttc acgtcgcggt caatcgtcgg gcggtcgatg ccgacaacgg 16501 ttagcggttg atcttcccgc acggccgccc aatcgcgggc actgccctgg ggatcggaat 16561 cgactaacag aacatcggcc ccggcgagtt gcagggcgcg ggctagatgg gttgcgatgg 16621 tcgtcttgcc tgacccgcct ttctggttaa gtacagcgat aaccttcatg cgttcccctt 16681 gcgtatttgt ttatttactc atcgcatcat atacgcagcg accgcatgac gcaagctgtt 16741 ttactcaaat acacatcacc tttttagacg gcggcgctcg gtttcttcag cggccaagct 16801 ggccggccag gccgccagct tggcatcaga caaaccggcc aggatttcat gcagccgcac 16861 ggttgagacg tgcgcgggcg gctcgaacac gtacccggcc gcgatcatct ccgcctcgat 16921 ctcttcggta atgaaaaacg gttcgtcctg gccgtcctgg tgcggtttca tgcttgttcc 16981 tcttggcgtt cattctcggc ggccgccagg gcgtcggcct cggtcaatgc gtcctcacgg 17041 aaggcaccgc gccgcctggc ctcggtgggc gtcacttcct cgctgcgctc aagtgcgcgg 17101 tacagggtcg agcgatgcac gccaagcagt gcagccgcct ctttcacggt gcggccttcc 17161 tggtcgatca gctcgcgggc gtgcgcgatc tgtgccgggg tgagggtagg gcgggggcca 17221 aacttcacgc ctcgggcctt ggcggcctcg cgcccgctcc gggtgcggtc gatgattagg 17281 gaacgctcga actcggcaat gccggcgaac acggtcaaca ccatgcggcc ggccggcgtg 17341 gtggtgtcgg cccacggctc tgccaggcta cgcaggcccg cgccggcctc ctggatgcgc 17401 tcggcaatgt ccagtaggtc gcgggtgctg cgggccaggc ggtctagcct ggtcactgtc 17461 acaacgtcgc cagggcgtag gtggtcaagc atcctggcca gctccgggcg gtcgcgcctg 17521 gtgccggtga tcttctcgga aaacagcttg gtgcagccgg ccgcgtgcag ttcggcccgt 17581 tggttggtca agtcctggtc gtcggtgctg acgcgggcat agcccagcag gccagcggcg 17641 gcgctcttgt tcatggcgta atgtctccgg ttctagtcgc aagtattcta ctttatgcga 17701 ctaaaacacg cgacaagaaa acgccaggaa aagggcaggg cggcagcctg tcgcgtaact 17761 taggacttgt gcgacatgtc gttttcagaa gacggctgca ctgaacgtca gaagccgact 17821 gcactatagc agcggagggg ttggatcaaa gtactttgat cccgagggga accctgtggt 17881 tggcatgcac atacaaatgg acgaacggat aaaccttttc acgccctttt aaatatccga 17941 ttattctaa //