LOCUS Exported 8494 bp ds-DNA circular SYN 26-JUL-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8494) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Jul 31, 2019 from SnapGene Viewer 4.3.8 https://www.snapgene.com FEATURES Location/Qualifiers source 1..8494 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 62..302 /label=U6 promoter primer_bind 107..131 /label=pORANGE sequencing primer FW misc_feature 307..328 /label=gRNA cloning site misc_RNA 329..404 /label=gRNA scaffold /note="in RNA form binds to Cas9" misc_feature 405..415 /label=Termination sequence /note="RNA polymerase will stop transcription at this position and fall off" misc_feature 489..558 /label=Multiple cloning site for the donor enhancer 559..844 /label=CMV enhancer primer_bind complement(567..586) /label=pORANGE sequencing primer RV promoter 846..1123 /label=chicken beta-actin promoter intron 1124..1351 /label=hybrid intron CDS 1369..1371 /codon_start=1 /label=START /translation="M" CDS 1372..1398 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 1402..1422 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 1432..5532 /codon_start=1 /product="Cas9 is from HITI construct. Suzuki et al., 2016" /label=Cas9 /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" CDS 5536..5556 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" polyA_signal 5602..5809 /label=bGH poly(A) signal repeat_region 5818..5958 /label=AAV2 ITR rep_origin 6033..6488 /direction=RIGHT /label=f1 ori /note="f1 stays for filamentous phages. It initiates a ""rolling circle"" replication in phage-infected bacteria and is useful if there is a need to produce vector as a single-stranded DNA" promoter 6770..6874 /label=AmpR promoter CDS 6875..7735 /codon_start=1 /label=AmpR /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 7906..8494 /direction=RIGHT /label=ori /note="Ori for replication in E.coli" ORIGIN 1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 301 acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa gttaaaataa 361 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc 421 tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga 481 caaatggctc tagaagcttg ctagcaattg tcgactcgag gatcctagga cgcgtcctag 541 gacgcgtcca gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 601 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca 661 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 721 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta 781 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 841 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 901 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 961 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 1021 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 1081 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 1141 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 1201 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct 1261 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct 1321 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat gtacccatac 1381 gatgttccag attacgcttc gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag 1441 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 1501 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 1561 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 1621 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 1681 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 1741 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1801 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1861 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1921 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 1981 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 2041 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 2101 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg 2161 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 2221 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 2281 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 2341 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 2401 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 2461 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 2521 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 2581 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 2641 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 2701 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 2761 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 2821 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 2881 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 2941 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 3001 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 3061 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 3121 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 3181 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 3241 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 3301 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 3361 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 3421 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 3481 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 3541 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 3601 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 3661 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 3721 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 3781 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 3841 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 3901 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 3961 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 4021 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 4081 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 4141 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 4201 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 4261 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 4321 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 4381 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 4441 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 4501 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 4561 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 4621 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 4681 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 4741 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 4801 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 4861 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 4921 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 4981 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 5041 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 5101 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 5161 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 5221 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 5281 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 5341 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 5401 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 5461 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 5521 ctgggaggcg acagccccaa gaagaagaga aaggtggagg ccagctaaga attctaacta 5581 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 5641 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 5701 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 5761 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg 5821 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 5881 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 5941 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 6001 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc 6061 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc 6121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 6181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 6241 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 6301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 6361 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg 6421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 6481 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 6541 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 6601 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 6661 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 6721 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 6781 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 6841 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 6901 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 6961 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 7021 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 7081 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 7141 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 7201 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 7261 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 7321 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 7381 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 7441 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 7501 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 7561 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc 7621 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 7681 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 7741 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 7801 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 7861 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 7921 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 7981 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 8041 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 8101 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 8161 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 8221 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 8281 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 8341 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 8401 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 8461 gtgatgctcg tcaggggggc ggagcctatg gaaa //