LOCUS Exported 16653 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR/Cas based plant genome editing and gene regulation; expresses 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance. ACCESSION . VERSION . KEYWORDS pHSE401 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16653) AUTHORS Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ TITLE A CRISPR/Cas9 toolkit for multiplex genome editing in plants. JOURNAL BMC Plant Biol. 2014 Nov 29;14(1):327. PUBMED 25432517 REFERENCE 2 (bases 1 to 16653) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16653 /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" CDS 977..1768 /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 1920..1995 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2609..2953 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" CDS 3106..3171 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 3178..3198 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3223..7323 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" RBS 7236..7244 /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 7324..7371 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(8028..8044) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8028..8044) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8041..8063) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8052..8068 /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(8076..8106) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8121..8142 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8333..9009 /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" CDS 9076..10101 /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" polyA_signal 10142..10316 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" misc_feature 10394..10418 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 10843..11637 /codon_start=1 /gene="aphA-3" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF" rep_origin 11724..12312 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 12213..12232 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 12466..12483 /label=L4440 /note="L4440 vector, forward primer" misc_feature 12498..12638 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(12564..12583) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 12724..12746 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 12982..13176 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(13242..14315) /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(14744..15373) /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 cttcgacttg ccttccgcac aatacatcat 301 ttcttcttag ctttttttct tcttcttcgt tcatacagtt tttttttgtt tatcagctta 361 cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt 421 atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt 481 gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga 541 atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata 601 ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa 661 gctgagttta tatacagcta gagtcgaagt agtgattggg agaccaaccc agtggacata 721 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag 781 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga 841 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt 901 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa 961 aacaaagtta aacatcatgg gggaagcggt gatcgccgaa gtatcgactc aactatcaga 1021 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg 1081 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac 1141 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc 1201 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga 1261 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa 1321 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct 1381 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga 1441 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc 1501 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta 1561 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg 1621 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga 1681 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg 1741 cgagatcacc aaggtagtcg gcaaataatg tctagctaga aattcgttca agccgacgcc 1801 gcttcgcggc gcggcttaac tcaagcgtta gatgcactaa gcacataatt gctcacagcc 1861 aaactatcag gtcaagtctg cttttattat ttttaagcgt gcataataag ccggtctcgg 1921 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg 1981 gcaccgagtc ggtgcttttt tttgcaaaat tttccagatc gatttcttct tcctctgttc 2041 ttcggcgttc aatttctggg gttttctctt cgttttctgt aactgaaacc taaaatttga 2101 cctaaaaaaa atctcaaata atatgattca gtggttttgt acttttcagt tagttgagtt 2161 ttgcagttcc gatgagataa accaataagc ttgcatgcct gcaggtcaac atggtggagc 2221 acgacacact tgtctactcc aaaaatatca aagatacagt ctcagaagac caaagggcaa 2281 ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta 2341 tctgtcactt tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt 2401 gcgataaagg aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac 2461 ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag 2521 tggattgatg tgataacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag 2581 atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 2641 acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 2701 aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 2761 ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 2821 acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg 2881 atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc 2941 atttggagag gacctcgacc tcaacacaac atatacaaaa caaacgaatc tcaagcaatc 3001 aagcattcta cttctattgc agcaatttaa atcatttctt ttaaagcaaa agcaattttc 3061 tgaaaatttt caccatttac gaacgatact cgagtaatct agatggatta caaggaccac 3121 gacggggatt acaaggacca cgacattgat tacaaggatg atgatgacaa gatggctccg 3181 aagaagaaga ggaaggttgg catccacggg gtgccagctg ctgacaagaa gtactcgatc 3241 ggcctcgata ttgggactaa ctctgttggc tgggccgtga tcaccgacga gtacaaggtg 3301 ccctcaaaga agttcaaggt cctgggcaac accgatcggc attccatcaa gaagaatctc 3361 attggcgctc tcctgttcga cagcggcgag acggctgagg ctacgcggct caagcgcacc 3421 gcccgcaggc ggtacacgcg caggaagaat cgcatctgct acctgcagga gattttctcc 3481 aacgagatgg cgaaggttga cgattctttc ttccacaggc tggaggagtc attcctcgtg 3541 gaggaggata agaagcacga gcggcatcca atcttcggca acattgtcga cgaggttgcc 3601 taccacgaga agtaccctac gatctaccat ctgcggaaga agctcgtgga ctccacagat 3661 aaggcggacc tccgcctgat ctacctcgct ctggcccaca tgattaagtt caggggccat 3721 ttcctgatcg agggggatct caacccggac aatagcgatg ttgacaagct gttcatccag 3781 ctcgtgcaga cgtacaacca gctcttcgag gagaacccca ttaatgcgtc aggcgtcgac 3841 gcgaaggcta tcctgtccgc taggctctcg aagtctcggc gcctcgagaa cctgatcgcc 3901 cagctgccgg gcgagaagaa gaacggcctg ttcgggaatc tcattgcgct cagcctgggg 3961 ctcacgccca acttcaagtc gaatttcgat ctcgctgagg acgccaagct gcagctctcc 4021 aaggacacat acgacgatga cctggataac ctcctggccc agatcggcga tcagtacgcg 4081 gacctgttcc tcgctgccaa gaatctgtcg gacgccatcc tcctgtctga tattctcagg 4141 gtgaacaccg agattacgaa ggctccgctc tcagcctcca tgatcaagcg ctacgacgag 4201 caccatcagg atctgaccct cctgaaggcg ctggtcaggc agcagctccc cgagaagtac 4261 aaggagatct tcttcgatca gtcgaagaac ggctacgctg ggtacattga cggcggggcc 4321 tctcaggagg agttctacaa gttcatcaag ccgattctgg agaagatgga cggcacggag 4381 gagctgctgg tgaagctcaa tcgcgaggac ctcctgagga agcagcggac attcgataac 4441 ggcagcatcc cacaccagat tcatctcggg gagctgcacg ctatcctgag gaggcaggag 4501 gacttctacc ctttcctcaa ggataaccgc gagaagatcg agaagattct gactttcagg 4561 atcccgtact acgtcggccc actcgctagg ggcaactccc gcttcgcttg gatgacccgc 4621 aagtcagagg agacgatcac gccgtggaac ttcgaggagg tggtcgacaa gggcgctagc 4681 gctcagtcgt tcatcgagag gatgacgaat ttcgacaaga acctgccaaa tgagaaggtg 4741 ctccctaagc actcgctcct gtacgagtac ttcacagtct acaacgagct gactaaggtg 4801 aagtatgtga ccgagggcat gaggaagccg gctttcctgt ctggggagca gaagaaggcc 4861 atcgtggacc tcctgttcaa gaccaaccgg aaggtcacgg ttaagcagct caaggaggac 4921 tacttcaaga agattgagtg cttcgattcg gtcgagatct ctggcgttga ggaccgcttc 4981 aacgcctccc tggggaccta ccacgatctc ctgaagatca ttaaggataa ggacttcctg 5041 gacaacgagg agaatgagga tatcctcgag gacattgtgc tgacactcac tctgttcgag 5101 gaccgggaga tgatcgagga gcgcctgaag acttacgccc atctcttcga tgacaaggtc 5161 atgaagcagc tcaagaggag gaggtacacc ggctggggga ggctgagcag gaagctcatc 5221 aacggcattc gggacaagca gtccgggaag acgatcctcg acttcctgaa gagcgatggc 5281 ttcgcgaacc gcaatttcat gcagctgatt cacgatgaca gcctcacatt caaggaggat 5341 atccagaagg ctcaggtgag cggccagggg gactcgctgc acgagcatat cgcgaacctc 5401 gctggctcgc cagctatcaa gaaggggatt ctgcagaccg tgaaggttgt ggacgagctg 5461 gtgaaggtca tgggcaggca caagcctgag aacatcgtca ttgagatggc ccgggagaat 5521 cagaccacgc agaagggcca gaagaactca cgcgagagga tgaagaggat cgaggagggc 5581 attaaggagc tggggtccca gatcctcaag gagcacccgg tggagaacac gcagctgcag 5641 aatgagaagc tctacctgta ctacctccag aatggccgcg atatgtatgt ggaccaggag 5701 ctggatatta acaggctcag cgattacgac gtcgatcata tcgttccaca gtcattcctg 5761 aaggatgact ccattgacaa caaggtcctc accaggtcgg acaagaaccg gggcaagtct 5821 gataatgttc cttcagagga ggtcgttaag aagatgaaga actactggcg ccagctcctg 5881 aatgccaagc tgatcacgca gcggaagttc gataacctca caaaggctga gaggggcggg 5941 ctctctgagc tggacaaggc gggcttcatc aagaggcagc tggtcgagac acggcagatc 6001 actaagcacg ttgcgcagat tctcgactca cggatgaaca ctaagtacga tgagaatgac 6061 aagctgatcc gcgaggtgaa ggtcatcacc ctgaagtcaa agctcgtctc cgacttcagg 6121 aaggatttcc agttctacaa ggttcgggag atcaacaatt accaccatgc ccatgacgcg 6181 tacctgaacg cggtggtcgg cacagctctg atcaagaagt acccaaagct cgagagcgag 6241 ttcgtgtacg gggactacaa ggtttacgat gtgaggaaga tgatcgccaa gtcggagcag 6301 gagattggca aggctaccgc caagtacttc ttctactcta acattatgaa tttcttcaag 6361 acagagatca ctctggccaa tggcgagatc cggaagcgcc ccctcatcga gacgaacggc 6421 gagacggggg agatcgtgtg ggacaagggc agggatttcg cgaccgtcag gaaggttctc 6481 tccatgccac aagtgaatat cgtcaagaag acagaggtcc agactggcgg gttctctaag 6541 gagtcaattc tgcctaagcg gaacagcgac aagctcatcg cccgcaagaa ggactgggat 6601 ccgaagaagt acggcgggtt cgacagcccc actgtggcct actcggtcct ggttgtggcg 6661 aaggttgaga agggcaagtc caagaagctc aagagcgtga aggagctgct ggggatcacg 6721 attatggagc gctccagctt cgagaagaac ccgatcgatt tcctggaggc gaagggctac 6781 aaggaggtga agaaggacct gatcattaag ctccccaagt actcactctt cgagctggag 6841 aacggcagga agcggatgct ggcttccgct ggcgagctgc agaaggggaa cgagctggct 6901 ctgccgtcca agtatgtgaa cttcctctac ctggcctccc actacgagaa gctcaagggc 6961 agccccgagg acaacgagca gaagcagctg ttcgtcgagc agcacaagca ttacctcgac 7021 gagatcattg agcagatttc cgagttctcc aagcgcgtga tcctggccga cgcgaatctg 7081 gataaggtcc tctccgcgta caacaagcac cgcgacaagc caatcaggga gcaggctgag 7141 aatatcattc atctcttcac cctgacgaac ctcggcgccc ctgctgcttt caagtacttc 7201 gacacaacta tcgatcgcaa gaggtacaca agcactaagg aggtcctgga cgcgaccctc 7261 atccaccagt cgattaccgg cctctacgag acgcgcatcg acctgtctca gctcgggggc 7321 gacaagcggc cagcggcgac gaagaaggcg gggcaggcga agaagaagaa gtgagctcag 7381 agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat 7441 tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc 7501 ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac 7561 tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc 7621 aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag 7681 atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag 7741 ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa 7801 tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt 7861 tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc 7921 attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta 7981 atgcatttta tgacttgcca attgattgac aacgaattcg taatcatgtc atagctgttt 8041 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 8101 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 8161 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 8221 gggagaggcg gtttgcgtat tggctagagc agcttgccaa catggtggag cacgacactc 8281 tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt 8341 ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact 8401 tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag 8461 gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 8521 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 8581 gtgaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca 8641 gaagaccaaa gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga 8701 ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc 8761 tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt 8821 ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 8881 acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa 8941 tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg 9001 acacgctgaa atcaccagtc tctctctaca aatctatctc tctcgagctt tcgcagatcc 9061 ggggggcaat gagatatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg 9121 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt 9181 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat 9241 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg 9301 gaagtgcttg acattgggga gtttagcgag agcctgacct attgcatctc ccgccgttca 9361 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct acaaccggtc 9421 gcggaggcta tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca 9481 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct 9541 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg 9601 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg 9661 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt 9721 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg 9781 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag 9841 cttgcaggat cgccacgact ccgggcgtat atgctccgca ttggtcttga ccaactctat 9901 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca 9961 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc 10021 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact 10081 cgtccgaggg caaagaaata gagtagatgc cgaccgggat ctgtcgatcg acaagctcga 10141 gtttctccat aataatgtgt gagtagttcc cagataaggg aattagggtt cctatagggt 10201 ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact 10261 tctatcaata aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatccccc 10321 gaattaattc ggcgttaatt cagtacatta aaaacgtccg caatgtgtta ttaagttgtc 10381 taagcgtcaa tttgtttaca ccacaatata tcctgccacc agccagccaa cagctccccg 10441 accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagtc cgggacggcg 10501 tcagcgggag agccgttgta aggcggcaga ctttgctcat gttaccgatg ctattcggaa 10561 gaacggcaac taagctgccg ggtttgaaac acggatgatc tcgcggaggg tagcatgttg 10621 attgtaacga tgacagagcg ttgctgcctg tgatcaccgc ggtttcaaaa tcggctccgt 10681 cgatactatg ttatacgcca actttgaaaa caactttgaa aaagctgttt tctggtattt 10741 aaggttttag aatgcaagga acagtgaatt ggagttcgtc ttgttataat tagcttcttg 10801 gggtatcttt aaatactgta gaaaagagga aggaaataat aaatggctaa aatgagaata 10861 tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac ggaaggaatg 10921 tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt aaaaatgacg 10981 gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat gatgctatgg 11041 ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga tggctggagc 11101 aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga agatgaacaa 11161 agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca ctccatcgac 11221 atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt ggattactta 11281 ctgaataacg atctggccga tgtggattgc gaaaactggg aagaagacac tccatttaaa 11341 gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga acttgtcttt 11401 tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt aagtggcttt 11461 attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt ctgcgtccgg 11521 tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga cttactgggg 11581 atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt gttttagtac 11641 ctagaatgca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 11701 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 11761 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 11821 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 11881 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 11941 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 12001 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 12061 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 12121 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 12181 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 12241 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 12301 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 12361 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 12421 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 12481 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 12541 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 12601 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg 12661 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 12721 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagg 12781 gtgccttgat gtgggcgccg gcggtcgagt ggcgacggcg cggcttgtcc gcgccctggt 12841 agattgcctg gccgtaggcc agccattttt gagcggccag cggccgcgat aggccgacgc 12901 gaagcggcgg ggcgtaggga gcgcagcgac cgaagggtag gcgctttttg cagctcttcg 12961 gctgtgcgct ggccagacag ttatgcacag gccaggcggg ttttaagagt tttaataagt 13021 tttaaagagt tttaggcgga aaaatcgcct tttttctctt ttatatcagt cacttacatg 13081 tgtgaccggt tcccaatgta cggctttggg ttcccaatgt acgggttccg gttcccaatg 13141 tacggctttg ggttcccaat gtacgtgcta tccacaggaa acagaccttt tcgacctttt 13201 tcccctgcta gggcaatttg ccctagcatc tgctccgtac attaggaacc ggcggatgct 13261 tcgccctcga tcaggttgcg gtagcgcatg actaggatcg ggccagcctg ccccgcctcc 13321 tccttcaaat cgtactccgg caggtcattt gacccgatca gcttgcgcac ggtgaaacag 13381 aacttcttga actctccggc gctgccactg cgttcgtaga tcgtcttgaa caaccatctg 13441 gcttctgcct tgcctgcggc gcggcgtgcc aggcggtaga gaaaacggcc gatgccggga 13501 tcgatcaaaa agtaatcggg gtgaaccgtc agcacgtccg ggttcttgcc ttctgtgatc 13561 tcgcggtaca tccaatcagc tagctcgatc tcgatgtact ccggccgccc ggtttcgctc 13621 tttacgatct tgtagcggct aatcaaggct tcaccctcgg ataccgtcac caggcggccg 13681 ttcttggcct tcttcgtacg ctgcatggca acgtgcgtgg tgtttaaccg aatgcaggtt 13741 tctaccaggt cgtctttctg ctttccgcca tcggctcgcc ggcagaactt gagtacgtcc 13801 gcaacgtgtg gacggaacac gcggccgggc ttgtctccct tcccttcccg gtatcggttc 13861 atggattcgg ttagatggga aaccgccatc agtaccaggt cgtaatccca cacactggcc 13921 atgccggccg gccctgcgga aacctctacg tgcccgtctg gaagctcgta gcggatcacc 13981 tcgccagctc gtcggtcacg cttcgacaga cggaaaacgg ccacgtccat gatgctgcga 14041 ctatcgcggg tgcccacgtc atagagcatc ggaacgaaaa aatctggttg ctcgtcgccc 14101 ttgggcggct tcctaatcga cggcgcaccg gctgccggcg gttgccggga ttctttgcgg 14161 attcgatcag cggccgcttg ccacgattca ccggggcgtg cttctgcctc gatgcgttgc 14221 cgctgggcgg cctgcgcggc cttcaacttc tccaccaggt catcacccag cgccgcgccg 14281 atttgtaccg ggccggatgg tttgcgaccg ctcacgccga ttcctcgggc ttgggggttc 14341 cagtgccatt gcagggccgg caggcaaccc agccgcttac gcctggccaa ccgcccgttc 14401 ctccacacat ggggcattcc acggcgtcgg tgcctggttg ttcttgattt tccatgccgc 14461 ctcctttagc cgctaaaatt catctactca tttattcatt tgctcattta ctctggtagc 14521 tgcgcgatgt attcagatag cagctcggta atggtcttgc cttggcgtac cgcgtacatc 14581 ttcagcttgg tgtgatcctc cgccggcaac tgaaagttga cccgcttcat ggctggcgtg 14641 tctgccaggc tggccaacgt tgcagccttg ctgctgcgtg cgctcggacg gccggcactt 14701 agcgtgtttg tgcttttgct cattttctct ttacctcatt aactcaaatg agttttgatt 14761 taatttcagc ggccagcgcc tggacctcgc gggcagcgtc gccctcgggt tctgattcaa 14821 gaacggttgt gccggcggcg gcagtgcctg ggtagctcac gcgctgcgtg atacgggact 14881 caagaatggg cagctcgtac ccggccagcg cctcggcaac ctcaccgccg atgcgcgtgc 14941 ctttgatcgc ccgcgacacg acaaaggccg cttgtagcct tccatccgtg acctcaatgc 15001 gctgcttaac cagctccacc aggtcggcgg tggcccatat gtcgtaaggg cttggctgca 15061 ccggaatcag cacgaagtcg gctgccttga tcgcggacac agccaagtcc gccgcctggg 15121 gcgctccgtc gatcactacg aagtcgcgcc ggccgatggc cttcacgtcg cggtcaatcg 15181 tcgggcggtc gatgccgaca acggttagcg gttgatcttc ccgcacggcc gcccaatcgc 15241 gggcactgcc ctggggatcg gaatcgacta acagaacatc ggccccggcg agttgcaggg 15301 cgcgggctag atgggttgcg atggtcgtct tgcctgaccc gcctttctgg ttaagtacag 15361 cgataacctt catgcgttcc ccttgcgtat ttgtttattt actcatcgca tcatatacgc 15421 agcgaccgca tgacgcaagc tgttttactc aaatacacat caccttttta gacggcggcg 15481 ctcggtttct tcagcggcca agctggccgg ccaggccgcc agcttggcat cagacaaacc 15541 ggccaggatt tcatgcagcc gcacggttga gacgtgcgcg ggcggctcga acacgtaccc 15601 ggccgcgatc atctccgcct cgatctcttc ggtaatgaaa aacggttcgt cctggccgtc 15661 ctggtgcggt ttcatgcttg ttcctcttgg cgttcattct cggcggccgc cagggcgtcg 15721 gcctcggtca atgcgtcctc acggaaggca ccgcgccgcc tggcctcggt gggcgtcact 15781 tcctcgctgc gctcaagtgc gcggtacagg gtcgagcgat gcacgccaag cagtgcagcc 15841 gcctctttca cggtgcggcc ttcctggtcg atcagctcgc gggcgtgcgc gatctgtgcc 15901 ggggtgaggg tagggcgggg gccaaacttc acgcctcggg ccttggcggc ctcgcgcccg 15961 ctccgggtgc ggtcgatgat tagggaacgc tcgaactcgg caatgccggc gaacacggtc 16021 aacaccatgc ggccggccgg cgtggtggtg tcggcccacg gctctgccag gctacgcagg 16081 cccgcgccgg cctcctggat gcgctcggca atgtccagta ggtcgcgggt gctgcgggcc 16141 aggcggtcta gcctggtcac tgtcacaacg tcgccagggc gtaggtggtc aagcatcctg 16201 gccagctccg ggcggtcgcg cctggtgccg gtgatcttct cggaaaacag cttggtgcag 16261 ccggccgcgt gcagttcggc ccgttggttg gtcaagtcct ggtcgtcggt gctgacgcgg 16321 gcatagccca gcaggccagc ggcggcgctc ttgttcatgg cgtaatgtct ccggttctag 16381 tcgcaagtat tctactttat gcgactaaaa cacgcgacaa gaaaacgcca ggaaaagggc 16441 agggcggcag cctgtcgcgt aacttaggac ttgtgcgaca tgtcgttttc agaagacggc 16501 tgcactgaac gtcagaagcc gactgcacta tagcagcgga ggggttggat caaagtactt 16561 tgatcccgag gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct 16621 tttcacgccc ttttaaatat ccgattattc taa //