LOCUS Exported 8401 bp ds-DNA circular SYN 14-MAY-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pYPQ173 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8401) AUTHORS . TITLE Direct Submission JOURNAL Exported May 14, 2018 from SnapGene 4.1.9 http://www.snapgene.com COMMENT LOCUS pNJB185_pNJB91_e 7579 bp ds-DNA circular 19-MAR-2014 SOURCE ORGANISM COMMENT This file is created by VectorDesigner COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers source 1..8401 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..139 /label=Spn promoter CDS 140..1150 /label=SpnR rep_origin 1217..1890 /direction=RIGHT /label=pUC origin terminator 2320..2363 /label=rrnB T1 terminatior primer_bind 2430..2445 /label=M13 (-20) forward primer misc_recomb 2462..2561 /label=attL1 CDS 2580..7016 /codon_start=1 /label=pcoCas9 /note="plant codon-optimized from Streptococcus pyogenes" /translation="MDYKDDDDKDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHE KYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRR QEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVSFC FYL*YIYNNYH*LVVI*YFKYFFQNKRM*YIAIAFL*FISVYILIYNFSNI*PKFVDVQ VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGDKRPAATKKAGQAKKK" misc_feature 2706..2708 /label=cyan misc_feature 5387 /label=cyan misc_feature 7017..7061 /label=5XGS linker /note="5XGS linker" exon 7062..7193 /label=VP64 /note="VP64" misc_feature 7194..7247 /label=T2A /note="T2A" misc_feature 7248..7637 /label=MS2 (At codon optimized) /note="MS2 (At codon optimized)" misc_feature 7638..7658 /label=NLS /note="NLS" misc_feature 7659..7688 /label=5X GS linker /note="5X GS linker" misc_feature 7689..7700 /label=BamHI-KpnI /note="BamHI-KpnI" exon 7701..7832 /label=VP64 /note="VP64" misc_feature 7701..7723 /label=VP64-F (SalI) /note="VP64-F (SalI)" primer_bind 7810..7832 /label=VP64-R (AatII) /note="VP64-R (AatII)" misc_feature 7845..7849 /label=AatII /note="AatII" misc_feature 7850..8127 /label=NOS term misc_recomb 8149..8272 /label=attR5 primer_bind 8313..8329 /label=M13 reverse primer promoter 8401 /label=Spn promoter ORIGIN 1 ctggaccagc caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg 61 cacaccgtgg aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa 121 gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag 181 cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca 241 gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt 301 atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat 361 gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga 421 gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg 481 cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac 541 aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga 601 gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta 661 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat 721 cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca 781 tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga 841 tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg 901 cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa 961 aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca 1021 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc 1081 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt 1141 cggcaaataa ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt 1201 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 1261 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 1321 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 1381 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 1441 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 1501 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 1561 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 1621 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 1681 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 1741 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 1801 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 1861 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 1921 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 1981 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 2041 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 2101 cagtgagcgc aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca 2161 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg 2221 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga 2281 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct 2341 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct 2401 tgcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc 2461 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca 2521 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tccgaattcg cccttcacca 2581 tggattacaa ggatgatgat gataaggatt acaaggatga tgatgataag atggctccaa 2641 agaagaagag aaaggttgga atccacggag ttccagctgc tgataagaag tactctatcg 2701 gacttgccat cggaaccaac tctgttggat gggctgttat caccgatgag tacaaggttc 2761 catctaagaa gttcaaggtt cttggaaaca ccgatagaca ctctatcaag aagaacctta 2821 tcggtgctct tcttttcgat tctggagaga ccgctgaggc taccagattg aagagaaccg 2881 ctagaagaag atacaccaga agaaagaaca gaatctgcta ccttcaggaa atcttctcta 2941 acgagatggc taaggttgat gattctttct tccacagact tgaggagtct ttccttgttg 3001 aggaggataa gaagcacgag agacacccaa tcttcggaaa catcgttgat gaggttgctt 3061 accacgagaa gtacccaacc atctaccacc ttagaaagaa gttggttgat tctaccgata 3121 aggctgatct tagacttatc taccttgctc ttgctcacat gatcaagttc agaggacact 3181 tccttatcga gggagacctt aacccagata actctgatgt tgataagttg ttcatccagc 3241 ttgttcagac ctacaaccag cttttcgagg agaacccaat caacgcttct ggagttgatg 3301 ctaaggctat cctttctgct agactttcta agtctcgtag acttgagaac cttatcgctc 3361 agcttccagg agagaagaag aacggacttt tcggaaacct tatcgctctt tctcttggac 3421 ttaccccaaa cttcaagtct aacttcgatc ttgctgagga tgctaagttg cagctttcta 3481 aggataccta cgatgatgat cttgataacc ttcttgctca gatcggagat cagtacgctg 3541 atcttttcct tgctgctaag aacctttctg atgctatcct tctttctgac atccttagag 3601 ttaacaccga gatcaccaag gctccacttt ctgcttctat gatcaagaga tacgatgagc 3661 accaccagga tcttaccctt ttgaaggctc ttgttagaca gcagcttcca gagaagtaca 3721 aggaaatctt cttcgatcag tctaagaacg gatacgctgg atacatcgat ggaggagctt 3781 ctcaggagga gttctacaag ttcatcaagc caatccttga gaagatggat ggaaccgagg 3841 agcttcttgt taagttgaac agagaggatc ttcttagaaa gcagagaacc ttcgataacg 3901 gatctatccc acaccagatc caccttggag agcttcacgc tatccttcgt agacaggagg 3961 atttctaccc attcttgaag gataacagag agaagatcga gaagatcctt accttcagaa 4021 tcccatacta cgttggacca cttgctagag gaaactctcg tttcgcttgg atgaccagaa 4081 agtctgagga gaccatcacc ccttggaact tcgaggaggt aagtttctgc ttctaccttt 4141 gatatatata taataattat cattaattag tagtaatata atatttcaaa tatttttttc 4201 aaaataaaag aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt 4261 taatttataa cttttctaat atatgaccaa aatttgttga tgtgcaggtt gttgataagg 4321 gagcttctgc tcagtctttc atcgagagaa tgaccaactt cgataagaac cttccaaacg 4381 agaaggttct tccaaagcac tctcttcttt acgagtactt caccgtttac aacgagctta 4441 ccaaggttaa gtacgttacc gagggaatga gaaagccagc tttcctttct ggagagcaga 4501 agaaggctat cgttgatctt cttttcaaga ccaacagaaa ggttaccgtt aagcagttga 4561 aggaggatta cttcaagaag atcgagtgct tcgattctgt tgaaatctct ggagttgagg 4621 atagattcaa cgcttctctt ggaacctacc acgatctttt gaagatcatc aaggataagg 4681 atttccttga taacgaggag aacgaggaca tccttgagga catcgttctt acccttaccc 4741 ttttcgagga tagagagatg atcgaggaga gactcaagac ctacgctcac cttttcgatg 4801 ataaggttat gaagcagttg aagagaagaa gatacaccgg atggggtaga ctttctcgta 4861 agttgatcaa cggaatcaga gataagcagt ctggaaagac catccttgat ttcttgaagt 4921 ctgatggatt cgctaacaga aacttcatgc agcttatcca cgatgattct cttaccttca 4981 aggaggacat ccagaaggct caggtttctg gacagggaga ttctcttcac gagcacatcg 5041 ctaaccttgc tggatctcca gctatcaaga agggaatcct tcagaccgtt aaggttgttg 5101 atgagcttgt taaggttatg ggtagacaca agccagagaa catcgttatc gagatggcta 5161 gagagaacca gaccacccag aagggacaga agaactctcg tgagagaatg aagagaatcg 5221 aggagggaat caaggagctt ggatctcaaa tcttgaagga gcacccagtt gagaacaccc 5281 agcttcagaa cgagaagttg tacctttact accttcagaa cggaagagat atgtacgttg 5341 atcaggagct tgacatcaac agactttctg attacgatgt tgatgccatc gttccacagt 5401 ctttcttgaa ggatgattct atcgataaca aggttcttac ccgttctgat aagaacagag 5461 gaaagtctga taacgttcca tctgaggagg ttgttaagaa gatgaagaac tactggagac 5521 agcttcttaa cgctaagttg atcacccaga gaaagttcga taaccttacc aaggctgaga 5581 gaggaggact ttctgagctt gataaggctg gattcatcaa gagacagctt gttgagacca 5641 gacagatcac caagcacgtt gctcagatcc ttgattctcg tatgaacacc aagtacgatg 5701 agaacgataa gttgatcaga gaggttaagg ttatcacctt gaagtctaag ttggtttctg 5761 atttcagaaa ggatttccag ttctacaagg ttagagagat caacaactac caccacgctc 5821 acgatgctta ccttaacgct gttgttggaa ccgctcttat caagaagtac ccaaagttgg 5881 agtctgagtt cgtttacgga gattacaagg tttacgatgt tagaaagatg atcgctaagt 5941 ctgagcagga gatcggaaag gctaccgcta agtacttctt ctactctaac atcatgaact 6001 tcttcaagac cgagatcacc cttgctaacg gagagatcag aaagagacca cttatcgaga 6061 ccaacggaga gaccggagag atcgtttggg ataagggaag agatttcgct accgttagaa 6121 aggttctttc tatgccacag gttaacatcg ttaagaaaac cgaggttcag accggaggat 6181 tctctaagga gtctatcctt ccaaagagaa actctgataa gttgatcgct agaaagaagg 6241 attgggaccc aaagaagtac ggaggattcg attctccaac cgttgcttac tctgttcttg 6301 ttgttgctaa ggttgagaag ggaaagtcta agaagttgaa gtctgttaag gagcttcttg 6361 gaatcaccat catggagcgt tcttctttcg agaagaaccc aatcgatttc cttgaggcta 6421 agggatacaa ggaggttaag aaggatctta tcatcaagtt gccaaagtac tctcttttcg 6481 agcttgagaa cggaagaaag agaatgcttg cttctgctgg agagcttcag aagggaaacg 6541 agcttgctct tccatctaag tacgttaact tcctttacct tgcttctcac tacgagaagt 6601 tgaagggatc tccagaggat aacgagcaga agcagctttt cgttgagcag cacaagcact 6661 accttgatga gatcatcgag caaatctctg agttctctaa gagagttatc cttgctgatg 6721 ctaaccttga taaggttctt tctgcttaca acaagcacag agataagcca atcagagagc 6781 aggctgagaa catcatccac cttttcaccc ttaccaacct tggtgctcca gctgctttca 6841 agtacttcga taccaccatc gatagaaaaa gatacacctc taccaaggag gttcttgatg 6901 ctacccttat ccaccagtct atcaccggac tttacgagac cagaatcgat ctttctcagc 6961 ttggaggaga taagagacca gctgctacca agaaggctgg acaggctaag aagaagaagg 7021 gagacggctc tggatcgggg tcgggttctg gctcagtcga cgatgctctt gacgattttg 7081 acctcgatat gctcgacgct cttgatgatt ttgatctcga catgctcgat gcacttgatg 7141 actttgacct tgacatgctc gacgcactcg atgacttcga cctcgacatg cttgagggca 7201 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg gcgtcaaatt 7261 tcacgcagtt tgttttggtt gataacggcg ggactggcga cgttacagta gctccatcaa 7321 attttgcgaa cggagtcgct gagtggatta gctcaaattc aaggtcccag gcctacaagg 7381 ttacctgttc tgttaggcag agttctgcgc aaaaaagaaa atataccatc aaggttgaag 7441 tccctaaagt tgcaacacaa acagtcggtg gtgttgagct ccctgtggca gcctggagat 7501 cttacttaaa catggagcta acaattccaa tattcgctac aaactctgat tgtgaactga 7561 ttgttaaggc gatgcaaggt ctcttgaaag atggaaaccc tataccgtcc gctatcgcag 7621 ctaacagcgg tatctatcct aagaagaaga ggaaggttgg ctctggatcg gggtcgggtt 7681 ctggctcagg atccggtacc gatgctcttg acgattttga cctcgatatg ctcgacgctc 7741 ttgatgattt tgatctcgac atgctcgatg cacttgatga ctttgacctt gacatgctcg 7801 acgcactcga tgacttcgac ctcgacatgc ttaagctttg ataggacgtc cgatcgttca 7861 aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 7921 atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 7981 tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 8041 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 8101 gatcgggaat tgatcccccc tcgacagctt ccggaaaggg cgaattcgca actttgtata 8161 caaaagttga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc 8221 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tgccatccag 8281 ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc 8341 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgcctcc 8401 t //