LOCUS Exported 16482 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Egg cell-specific promoter-controlled expression 3×FLAG-NLS-zCas9-NLS and two sgRNAs targeting three genes: ETC2, TRY, and CPC. ACCESSION . VERSION . KEYWORDS pHEE2E-TRI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16482) AUTHORS Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ TITLE Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. JOURNAL Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0. PUBMED 26193878 REFERENCE 2 (bases 1 to 16482) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16482 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(19..38) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 179..201 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 437..631 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(697..1770) /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(2199..2828) /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" misc_feature 4129..4153 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" primer_bind 4341..4363 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4355..4372 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 4356..4372 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_RNA 4826..4901 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_RNA 5398..5473 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 7043..7108 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 7115..7135 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7160..11260 /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 11173..11181 /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 11261..11308 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(11965..11981) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11965..11981) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11978..12000) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11989..12005 /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(12013..12043) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12058..12079 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 12270..12946 /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" primer_bind 12883..12903 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 13013..14038 /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 14079..14253 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" misc_feature 14331..14355 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 14780..15574 /codon_start=1 /gene="aphA-3" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF" rep_origin 15661..16249 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 16150..16169 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 16403..16420 /label=L4440 /note="L4440 vector, forward primer" misc_feature 16435..93 /label=bom /note="basis of mobility region from pBR322" ORIGIN 1 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact 61 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac 121 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 181 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagggtgcc 241 ttgatgtggg cgccggcggt cgagtggcga cggcgcggct tgtccgcgcc ctggtagatt 301 gcctggccgt aggccagcca tttttgagcg gccagcggcc gcgataggcc gacgcgaagc 361 ggcggggcgt agggagcgca gcgaccgaag ggtaggcgct ttttgcagct cttcggctgt 421 gcgctggcca gacagttatg cacaggccag gcgggtttta agagttttaa taagttttaa 481 agagttttag gcggaaaaat cgcctttttt ctcttttata tcagtcactt acatgtgtga 541 ccggttccca atgtacggct ttgggttccc aatgtacggg ttccggttcc caatgtacgg 601 ctttgggttc ccaatgtacg tgctatccac aggaaacaga ccttttcgac ctttttcccc 661 tgctagggca atttgcccta gcatctgctc cgtacattag gaaccggcgg atgcttcgcc 721 ctcgatcagg ttgcggtagc gcatgactag gatcgggcca gcctgccccg cctcctcctt 781 caaatcgtac tccggcaggt catttgaccc gatcagcttg cgcacggtga aacagaactt 841 cttgaactct ccggcgctgc cactgcgttc gtagatcgtc ttgaacaacc atctggcttc 901 tgccttgcct gcggcgcggc gtgccaggcg gtagagaaaa cggccgatgc cgggatcgat 961 caaaaagtaa tcggggtgaa ccgtcagcac gtccgggttc ttgccttctg tgatctcgcg 1021 gtacatccaa tcagctagct cgatctcgat gtactccggc cgcccggttt cgctctttac 1081 gatcttgtag cggctaatca aggcttcacc ctcggatacc gtcaccaggc ggccgttctt 1141 ggccttcttc gtacgctgca tggcaacgtg cgtggtgttt aaccgaatgc aggtttctac 1201 caggtcgtct ttctgctttc cgccatcggc tcgccggcag aacttgagta cgtccgcaac 1261 gtgtggacgg aacacgcggc cgggcttgtc tcccttccct tcccggtatc ggttcatgga 1321 ttcggttaga tgggaaaccg ccatcagtac caggtcgtaa tcccacacac tggccatgcc 1381 ggccggccct gcggaaacct ctacgtgccc gtctggaagc tcgtagcgga tcacctcgcc 1441 agctcgtcgg tcacgcttcg acagacggaa aacggccacg tccatgatgc tgcgactatc 1501 gcgggtgccc acgtcataga gcatcggaac gaaaaaatct ggttgctcgt cgcccttggg 1561 cggcttccta atcgacggcg caccggctgc cggcggttgc cgggattctt tgcggattcg 1621 atcagcggcc gcttgccacg attcaccggg gcgtgcttct gcctcgatgc gttgccgctg 1681 ggcggcctgc gcggccttca acttctccac caggtcatca cccagcgccg cgccgatttg 1741 taccgggccg gatggtttgc gaccgctcac gccgattcct cgggcttggg ggttccagtg 1801 ccattgcagg gccggcaggc aacccagccg cttacgcctg gccaaccgcc cgttcctcca 1861 cacatggggc attccacggc gtcggtgcct ggttgttctt gattttccat gccgcctcct 1921 ttagccgcta aaattcatct actcatttat tcatttgctc atttactctg gtagctgcgc 1981 gatgtattca gatagcagct cggtaatggt cttgccttgg cgtaccgcgt acatcttcag 2041 cttggtgtga tcctccgccg gcaactgaaa gttgacccgc ttcatggctg gcgtgtctgc 2101 caggctggcc aacgttgcag ccttgctgct gcgtgcgctc ggacggccgg cacttagcgt 2161 gtttgtgctt ttgctcattt tctctttacc tcattaactc aaatgagttt tgatttaatt 2221 tcagcggcca gcgcctggac ctcgcgggca gcgtcgccct cgggttctga ttcaagaacg 2281 gttgtgccgg cggcggcagt gcctgggtag ctcacgcgct gcgtgatacg ggactcaaga 2341 atgggcagct cgtacccggc cagcgcctcg gcaacctcac cgccgatgcg cgtgcctttg 2401 atcgcccgcg acacgacaaa ggccgcttgt agccttccat ccgtgacctc aatgcgctgc 2461 ttaaccagct ccaccaggtc ggcggtggcc catatgtcgt aagggcttgg ctgcaccgga 2521 atcagcacga agtcggctgc cttgatcgcg gacacagcca agtccgccgc ctggggcgct 2581 ccgtcgatca ctacgaagtc gcgccggccg atggccttca cgtcgcggtc aatcgtcggg 2641 cggtcgatgc cgacaacggt tagcggttga tcttcccgca cggccgccca atcgcgggca 2701 ctgccctggg gatcggaatc gactaacaga acatcggccc cggcgagttg cagggcgcgg 2761 gctagatggg ttgcgatggt cgtcttgcct gacccgcctt tctggttaag tacagcgata 2821 accttcatgc gttccccttg cgtatttgtt tatttactca tcgcatcata tacgcagcga 2881 ccgcatgacg caagctgttt tactcaaata cacatcacct ttttagacgg cggcgctcgg 2941 tttcttcagc ggccaagctg gccggccagg ccgccagctt ggcatcagac aaaccggcca 3001 ggatttcatg cagccgcacg gttgagacgt gcgcgggcgg ctcgaacacg tacccggccg 3061 cgatcatctc cgcctcgatc tcttcggtaa tgaaaaacgg ttcgtcctgg ccgtcctggt 3121 gcggtttcat gcttgttcct cttggcgttc attctcggcg gccgccaggg cgtcggcctc 3181 ggtcaatgcg tcctcacgga aggcaccgcg ccgcctggcc tcggtgggcg tcacttcctc 3241 gctgcgctca agtgcgcggt acagggtcga gcgatgcacg ccaagcagtg cagccgcctc 3301 tttcacggtg cggccttcct ggtcgatcag ctcgcgggcg tgcgcgatct gtgccggggt 3361 gagggtaggg cgggggccaa acttcacgcc tcgggccttg gcggcctcgc gcccgctccg 3421 ggtgcggtcg atgattaggg aacgctcgaa ctcggcaatg ccggcgaaca cggtcaacac 3481 catgcggccg gccggcgtgg tggtgtcggc ccacggctct gccaggctac gcaggcccgc 3541 gccggcctcc tggatgcgct cggcaatgtc cagtaggtcg cgggtgctgc gggccaggcg 3601 gtctagcctg gtcactgtca caacgtcgcc agggcgtagg tggtcaagca tcctggccag 3661 ctccgggcgg tcgcgcctgg tgccggtgat cttctcggaa aacagcttgg tgcagccggc 3721 cgcgtgcagt tcggcccgtt ggttggtcaa gtcctggtcg tcggtgctga cgcgggcata 3781 gcccagcagg ccagcggcgg cgctcttgtt catggcgtaa tgtctccggt tctagtcgca 3841 agtattctac tttatgcgac taaaacacgc gacaagaaaa cgccaggaaa agggcagggc 3901 ggcagcctgt cgcgtaactt aggacttgtg cgacatgtcg ttttcagaag acggctgcac 3961 tgaacgtcag aagccgactg cactatagca gcggaggggt tggatcaaag tactttgatc 4021 ccgaggggaa ccctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca 4081 cgccctttta aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca 4141 atatatcctg tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgatccaa 4201 gctcaagctg ctctagcatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 4261 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 4321 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 4381 tcgacttgcc ttccgcacaa tacatcattt cttcttagct ttttttcttc ttcttcgttc 4441 atacagtttt tttttgttta tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc 4501 tttttaactt tccattcgga gtttttgtat cttgtttcat agtttgtccc aggattagaa 4561 tgattaggca tcgaaccttc aagaatttga ttgaataaaa catcttcatt cttaagatat 4621 gaagataatc ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat 4681 gggaaagaac aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag 4741 tcccacatcg cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag 4801 tgattgaata tctctctatc tcctcgtttt agagctagaa atagcaagtt aaaataaggc 4861 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttg caaaattttc 4921 cagatcgatt tcttcttcct ctgttcttcg gcgttcaatt tctggggttt tctcttcgtt 4981 ttctgtaact gaaacctaaa atttgaccta aaaaaatctc aaataatatg attcagtggt 5041 tttgtacttt tcagttagtt gagttttgca gttccgatga gataaaccaa tattaatcca 5101 aactactgca gcctgacaga caaatgagga tgcaaacaat tttaaagttt atctaacgct 5161 agctgttttg tttcttctct ctggtgcacc aacgacggcg ttttctcaat cataaagagg 5221 cttgttttac ttaaggccaa taatgttgat ggatcgaaag aagagggctt ttaataaacg 5281 agcccgttta agctgtaaac gatgtcaaaa acatcccaca tcgttcagtt gaaaatagaa 5341 gctctgttta tatattggta gagtcgacta agagattgga agtgagtagc atcgaatgtt 5401 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 5461 accgagtcgg tgcttttttt tgcaaaattt tccagatcga tttcttcttc ctctgttctt 5521 cggcgttcaa tttctggggt tttctcttcg ttttctgtaa ctgaaaccta aaatttgacc 5581 taaaaaaaat ctcaaataat atgattcagt ggttttgtac ttttcagtta gttgagtttt 5641 gcagttccga tgagataaac caataccatg gttatactag tgaataaaag catttgcgtt 5701 tggtttatca ttgcgtttat acaaggacag agatccactg agctggaata gcttaaaacc 5761 attatcagaa caaaataaac cattttttgt taagaatcag agcatagtaa acaacagaaa 5821 caacctaaga gaggtaactt gtccaagaag atagctaatt atatctattt tataaaagtt 5881 atcatagttt gtaagtcaca aaagatgcaa ataacagaga aactaggaga cttgagaata 5941 tacattcttg tatatttgta ttcgagattg tgaaaatttg accataagtt taaattctta 6001 aaaagatata tctgatctag gtgatggtta tagactgtaa ttttaccaca tgtttaatga 6061 tggatagtga cacacatgac acatcgacaa cactatagca tcttatttag attacaacat 6121 gaaatttttc tgtaatacat gtctttgtac ataatttaaa agtaattcct aagaaatata 6181 tttatacaag gagtttaaag aaaacatagc ataaagttca atgagtagta aaaaccatat 6241 acagtatata gcataaagtt caatgagttt attacaaaag cattggttca ctttctgtaa 6301 cacgacgtta aaccttcgtc tccaatagga gcgctactga ttcaacatgc caatatatac 6361 taaatacgtt tctacagtca aatgctttaa cgtttcatga ttaagtgact atttaccgtc 6421 aatcctttcc cattcctccc actaatccaa ctttttaatt actcttaaat caccactaag 6481 ctagtaacgc ctatcatgaa ttagctctac taaatctagc aacctttcaa atttgcagta 6541 ttgcaggtgt ctctgtgtct ttaaaatagt tgccttatga tttcttcggt ttcaagatga 6601 tcaaatagtt atagatttca tgctcacaca tgctcattag atgtgtacat actttactta 6661 cccaaatcta ttttctcgca aagattttga tggtaaagct gatttggttc tattgaacta 6721 aatcaaacga gtttcagact gagtgattct aatccggccc attagcccct aaacagaccc 6781 actaattacg cagcttttaa tagagtaatt acacctagtt tacccactaa accactaagc 6841 actaattatc tcacaatcta atgagcttcc ctcgtaatta cttgggcttt cactctacca 6901 tttatttgta acagtcaagt ctctactgtc tctatataaa ctctctaaag ttaacacaca 6961 attctcatca caaacaaatc aaccaaagca acttctactc tttcttcttt cgaccttatc 7021 aatctgttga gaaatctaga tggattacaa ggaccacgac ggggattaca aggaccacga 7081 cattgattac aaggatgatg atgacaagat ggctccgaag aagaagagga aggttggcat 7141 ccacggggtg ccagctgctg acaagaagta ctcgatcggc ctcgatattg ggactaactc 7201 tgttggctgg gccgtgatca ccgacgagta caaggtgccc tcaaagaagt tcaaggtcct 7261 gggcaacacc gatcggcatt ccatcaagaa gaatctcatt ggcgctctcc tgttcgacag 7321 cggcgagacg gctgaggcta cgcggctcaa gcgcaccgcc cgcaggcggt acacgcgcag 7381 gaagaatcgc atctgctacc tgcaggagat tttctccaac gagatggcga aggttgacga 7441 ttctttcttc cacaggctgg aggagtcatt cctcgtggag gaggataaga agcacgagcg 7501 gcatccaatc ttcggcaaca ttgtcgacga ggttgcctac cacgagaagt accctacgat 7561 ctaccatctg cggaagaagc tcgtggactc cacagataag gcggacctcc gcctgatcta 7621 cctcgctctg gcccacatga ttaagttcag gggccatttc ctgatcgagg gggatctcaa 7681 cccggacaat agcgatgttg acaagctgtt catccagctc gtgcagacgt acaaccagct 7741 cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc tgtccgctag 7801 gctctcgaag tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg agaagaagaa 7861 cggcctgttc gggaatctca ttgcgctcag cctggggctc acgcccaact tcaagtcgaa 7921 tttcgatctc gctgaggacg ccaagctgca gctctccaag gacacatacg acgatgacct 7981 ggataacctc ctggcccaga tcggcgatca gtacgcggac ctgttcctcg ctgccaagaa 8041 tctgtcggac gccatcctcc tgtctgatat tctcagggtg aacaccgaga ttacgaaggc 8101 tccgctctca gcctccatga tcaagcgcta cgacgagcac catcaggatc tgaccctcct 8161 gaaggcgctg gtcaggcagc agctccccga gaagtacaag gagatcttct tcgatcagtc 8221 gaagaacggc tacgctgggt acattgacgg cggggcctct caggaggagt tctacaagtt 8281 catcaagccg attctggaga agatggacgg cacggaggag ctgctggtga agctcaatcg 8341 cgaggacctc ctgaggaagc agcggacatt cgataacggc agcatcccac accagattca 8401 tctcggggag ctgcacgcta tcctgaggag gcaggaggac ttctaccctt tcctcaagga 8461 taaccgcgag aagatcgaga agattctgac tttcaggatc ccgtactacg tcggcccact 8521 cgctaggggc aactcccgct tcgcttggat gacccgcaag tcagaggaga cgatcacgcc 8581 gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca tcgagaggat 8641 gacgaatttc gacaagaacc tgccaaatga gaaggtgctc cctaagcact cgctcctgta 8701 cgagtacttc acagtctaca acgagctgac taaggtgaag tatgtgaccg agggcatgag 8761 gaagccggct ttcctgtctg gggagcagaa gaaggccatc gtggacctcc tgttcaagac 8821 caaccggaag gtcacggtta agcagctcaa ggaggactac ttcaagaaga ttgagtgctt 8881 cgattcggtc gagatctctg gcgttgagga ccgcttcaac gcctccctgg ggacctacca 8941 cgatctcctg aagatcatta aggataagga cttcctggac aacgaggaga atgaggatat 9001 cctcgaggac attgtgctga cactcactct gttcgaggac cgggagatga tcgaggagcg 9061 cctgaagact tacgcccatc tcttcgatga caaggtcatg aagcagctca agaggaggag 9121 gtacaccggc tgggggaggc tgagcaggaa gctcatcaac ggcattcggg acaagcagtc 9181 cgggaagacg atcctcgact tcctgaagag cgatggcttc gcgaaccgca atttcatgca 9241 gctgattcac gatgacagcc tcacattcaa ggaggatatc cagaaggctc aggtgagcgg 9301 ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcgccag ctatcaagaa 9361 ggggattctg cagaccgtga aggttgtgga cgagctggtg aaggtcatgg gcaggcacaa 9421 gcctgagaac atcgtcattg agatggcccg ggagaatcag accacgcaga agggccagaa 9481 gaactcacgc gagaggatga agaggatcga ggagggcatt aaggagctgg ggtcccagat 9541 cctcaaggag cacccggtgg agaacacgca gctgcagaat gagaagctct acctgtacta 9601 cctccagaat ggccgcgata tgtatgtgga ccaggagctg gatattaaca ggctcagcga 9661 ttacgacgtc gatcatatcg ttccacagtc attcctgaag gatgactcca ttgacaacaa 9721 ggtcctcacc aggtcggaca agaaccgggg caagtctgat aatgttcctt cagaggaggt 9781 cgttaagaag atgaagaact actggcgcca gctcctgaat gccaagctga tcacgcagcg 9841 gaagttcgat aacctcacaa aggctgagag gggcgggctc tctgagctgg acaaggcggg 9901 cttcatcaag aggcagctgg tcgagacacg gcagatcact aagcacgttg cgcagattct 9961 cgactcacgg atgaacacta agtacgatga gaatgacaag ctgatccgcg aggtgaaggt 10021 catcaccctg aagtcaaagc tcgtctccga cttcaggaag gatttccagt tctacaaggt 10081 tcgggagatc aacaattacc accatgccca tgacgcgtac ctgaacgcgg tggtcggcac 10141 agctctgatc aagaagtacc caaagctcga gagcgagttc gtgtacgggg actacaaggt 10201 ttacgatgtg aggaagatga tcgccaagtc ggagcaggag attggcaagg ctaccgccaa 10261 gtacttcttc tactctaaca ttatgaattt cttcaagaca gagatcactc tggccaatgg 10321 cgagatccgg aagcgccccc tcatcgagac gaacggcgag acgggggaga tcgtgtggga 10381 caagggcagg gatttcgcga ccgtcaggaa ggttctctcc atgccacaag tgaatatcgt 10441 caagaagaca gaggtccaga ctggcgggtt ctctaaggag tcaattctgc ctaagcggaa 10501 cagcgacaag ctcatcgccc gcaagaagga ctgggatccg aagaagtacg gcgggttcga 10561 cagccccact gtggcctact cggtcctggt tgtggcgaag gttgagaagg gcaagtccaa 10621 gaagctcaag agcgtgaagg agctgctggg gatcacgatt atggagcgct ccagcttcga 10681 gaagaacccg atcgatttcc tggaggcgaa gggctacaag gaggtgaaga aggacctgat 10741 cattaagctc cccaagtact cactcttcga gctggagaac ggcaggaagc ggatgctggc 10801 ttccgctggc gagctgcaga aggggaacga gctggctctg ccgtccaagt atgtgaactt 10861 cctctacctg gcctcccact acgagaagct caagggcagc cccgaggaca acgagcagaa 10921 gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcattgagc agatttccga 10981 gttctccaag cgcgtgatcc tggccgacgc gaatctggat aaggtcctct ccgcgtacaa 11041 caagcaccgc gacaagccaa tcagggagca ggctgagaat atcattcatc tcttcaccct 11101 gacgaacctc ggcgcccctg ctgctttcaa gtacttcgac acaactatcg atcgcaagag 11161 gtacacaagc actaaggagg tcctggacgc gaccctcatc caccagtcga ttaccggcct 11221 ctacgagacg cgcatcgacc tgtctcagct cgggggcgac aagcggccag cggcgacgaa 11281 gaaggcgggg caggcgaaga agaagaagtg agctcagagc tttcgttcgt atcatcggtt 11341 tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc gcacacacca gaatcctact 11401 gagtttgagt attatggcat tgggaaaact gtttttcttg taccatttgt tgtgcttgta 11461 atttactgtg ttttttattc ggttttcgct atcgaactgt gaaatggaaa tggatggaga 11521 agagttaatg aatgatatgg tccttttgtt cattctcaaa ttaatattat ttgttttttc 11581 tcttatttgt tgtgtgttga atttgaaatt ataagagata tgcaaacatt ttgttttgag 11641 taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta atatgaggag taaaacactt 11701 gtagttgtac cattatgctt attcactagg caacaaatat attttcagac ctagaaaagc 11761 tgcaaatgtt actgaataca agtatgtcct cttgtgtttt agacatttat gaactttcct 11821 ttatgtaatt ttccagaatc cttgtcagat tctaatcatt gctttataat tatagttata 11881 ctcatggatt tgtagttgag tatgaaaata ttttttaatg cattttatga cttgccaatt 11941 gattgacaac gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct 12001 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 12061 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 12121 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 12181 ctagagcagc ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 12241 gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 12301 aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 12361 gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 12421 tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 12481 gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg aacatggtgg agcacgacac 12541 tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac 12601 ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca 12661 cttcatcaaa aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa 12721 aggaaaggct atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 12781 cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 12841 atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc 12901 ttcctctata taaggaagtt catttcattt ggagaggaca cgctgaaatc accagtctct 12961 ctctacaaat ctatctctct cgagctttcg cagatccggg gggcaatgag atatgaaaaa 13021 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 13081 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 13141 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 13201 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggagtt 13261 tagcgagagc ctgacctatt gcatctcccg ccgttcacag ggtgtcacgt tgcaagacct 13321 gcctgaaacc gaactgcccg ctgttctaca accggtcgcg gaggctatgg atgcgatcgc 13381 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 13441 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca 13501 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 13561 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 13621 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 13681 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 13741 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cacgactccg 13801 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 13861 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 13921 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 13981 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa agaaatagag 14041 tagatgccga ccgggatctg tcgatcgaca agctcgagtt tctccataat aatgtgtgag 14101 tagttcccag ataagggaat tagggttcct atagggtttc gctcatgtgt tgagcatata 14161 agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc 14221 ctaaaaccaa aatccagtac taaaatccag atcccccgaa ttaattcggc gttaattcag 14281 tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca 14341 caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca 14401 ccactcgata caggcagccc atcagtccgg gacggcgtca gcgggagagc cgttgtaagg 14461 cggcagactt tgctcatgtt accgatgcta ttcggaagaa cggcaactaa gctgccgggt 14521 ttgaaacacg gatgatctcg cggagggtag catgttgatt gtaacgatga cagagcgttg 14581 ctgcctgtga tcaccgcggt ttcaaaatcg gctccgtcga tactatgtta tacgccaact 14641 ttgaaaacaa ctttgaaaaa gctgttttct ggtatttaag gttttagaat gcaaggaaca 14701 gtgaattgga gttcgtcttg ttataattag cttcttgggg tatctttaaa tactgtagaa 14761 aagaggaagg aaataataaa tggctaaaat gagaatatca ccggaattga aaaaactgat 14821 cgaaaaatac cgctgcgtaa aagatacgga aggaatgtct cctgctaagg tatataagct 14881 ggtgggagaa aatgaaaacc tatatttaaa aatgacggac agccggtata aagggaccac 14941 ctatgatgtg gaacgggaaa aggacatgat gctatggctg gaaggaaagc tgcctgttcc 15001 aaaggtcctg cactttgaac ggcatgatgg ctggagcaat ctgctcatga gtgaggccga 15061 tggcgtcctt tgctcggaag agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct 15121 gtatgcggag tgcatcaggc tctttcactc catcgacata tcggattgtc cctatacgaa 15181 tagcttagac agccgcttag ccgaattgga ttacttactg aataacgatc tggccgatgt 15241 ggattgcgaa aactgggaag aagacactcc atttaaagat ccgcgcgagc tgtatgattt 15301 tttaaagacg gaaaagcccg aagaggaact tgtcttttcc cacggcgacc tgggagacag 15361 caacatcttt gtgaaagatg gcaaagtaag tggctttatt gatcttggga gaagcggcag 15421 ggcggacaag tggtatgaca ttgccttctg cgtccggtcg atcagggagg atatcgggga 15481 agaacagtat gtcgagctat tttttgactt actggggatc aagcctgatt gggagaaaat 15541 aaaatattat attttactgg atgaattgtt ttagtaccta gaatgcatga ccaaaatccc 15601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 15661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 15721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 15781 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 15841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 15901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 15961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 16021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 16081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 16141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 16201 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 16261 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 16321 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 16381 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 16441 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cg //