LOCUS Exported 9257 bp ds-DNA circular SYN 23-JUL-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9257) 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..9257 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 39..279 /label=U6 promoter primer_bind 84..108 /label=pORANGE sequencing primer FW misc_feature 288..307 /label=Target sequence misc_RNA 308..383 /label=gRNA scaffold misc_feature 384..394 /label=Termination sequence misc_feature 479..481 /label=PAM misc_feature 482..501 /label=Target sequence CDS 502..510 /codon_start=1 /label=Linker /translation="ASG" CDS 511..1224 /codon_start=1 /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 1225..1230 /codon_start=1 /label=Linker /translation="SA" misc_feature 1232..1234 /label=PAM misc_feature 1235..1254 /label=Target sequence enhancer 1299..1584 /label=CMV enhancer primer_bind complement(1307..1326) /label=pORANGE sequencing primer RV promoter 1586..1863 /label=chicken beta-actin promoter intron 1864..2091 /label=hybrid intron misc_feature 2109..2111 /label=START CDS 2112..2138 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 2142..2162 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 2172..6272 /codon_start=1 /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 6276..6296 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" polyA_signal 6342..6549 /label=bGH poly(A) signal repeat_region 6558..6698 /label=AAV2 ITR rep_origin 6773..7228 /direction=RIGHT /label=f1 ori promoter 7510..7614 /label=AmpR promoter CDS 7615..8475 /codon_start=1 /label=AmpR /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8646..9234 /direction=RIGHT /label=ori ORIGIN 1 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc 61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgac catgtaccgg 301 gacccgggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg 361 aaaaagtggc accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata 421 aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac aaatggctct agaagcttcc 481 cccgggtccc ggtacatggt cgctagcgga gtgagcaagg gcgaggagct gttcaccggg 541 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 601 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 661 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 721 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 781 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 841 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 901 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 961 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1021 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1081 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1141 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1201 ctcggcatgg acgagctgta caagagcgct tccgccgggt cccggtacat ggtcctcgag 1261 gatcctagga cgcgtcctag gacgcgtcca gaggtacccg ttacataact tacggtaaat 1321 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata 1381 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 1441 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 1501 gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1561 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc 1621 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 1681 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg 1741 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag 1801 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg 1861 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc 1921 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct 1981 cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg 2041 gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg 2101 gtgccaccat gtacccatac gatgttccag attacgcttc gccgaagaaa aagcgcaagg 2161 tcgaagcgtc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct 2221 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 2281 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa 2341 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc 2401 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct 2461 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca 2521 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 2581 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 2641 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 2701 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 2761 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 2821 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 2881 tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 2941 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 3001 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 3061 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 3121 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc 3181 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 3241 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 3301 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 3361 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag 3421 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 3481 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 3541 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 3601 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 3661 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 3721 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 3781 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 3841 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 3901 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 3961 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 4021 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 4081 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 4141 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 4201 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 4261 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 4321 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 4381 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 4441 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 4501 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 4561 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 4621 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 4681 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 4741 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 4801 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 4861 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 4921 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 4981 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 5041 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 5101 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 5161 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 5221 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 5281 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 5341 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 5401 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 5461 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 5521 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 5581 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 5641 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 5701 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 5761 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 5821 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 5881 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 5941 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 6001 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 6061 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 6121 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 6181 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 6241 cacggatcga cctgtctcag ctgggaggcg acagccccaa gaagaagaga aaggtggagg 6301 ccagctaaga attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 6361 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 6421 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6481 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 6541 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 6601 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 6661 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 6721 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 6781 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 6841 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 6901 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6961 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 7021 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 7081 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt 7141 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 7201 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 7261 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 7321 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 7381 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 7441 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 7501 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 7561 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 7621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 7681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 7741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 7801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 7861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 7921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 7981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 8041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 8101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 8161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 8221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 8281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt 8341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 8401 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 8461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 8521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 8581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 8641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 8701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 8761 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 8821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 8881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 8941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 9001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 9061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 9121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 9181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 9241 agcaacgcgg ccttttt //