LOCUS Exported 7667 bp ds-DNA circular SYN 25-OCT-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 7667) AUTHORS Zhenya TITLE Direct Submission JOURNAL Exported Friday, Oct 25, 2019 from SnapGene 4.3.6 https://www.snapgene.com FEATURES Location/Qualifiers source 1..7667 /organism="recombinant plasmid" /mol_type="other DNA" misc_feature 1..130 /label=AAV_ITR? enhancer 154..533 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 534..737 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 765..830 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 999..4253 /codon_start=1 /product="http://www.uniprot.org/uniprot/J7RUA5" /label=S.a. Cas9 ORF /translation="PKKKRKVGIHGVPAAKRNYILGLAIGITSVGYGIIDYETRDVIDA GVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKY VAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETR RTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVI TRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNL KVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDF ILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNE RIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS VSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKT KKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE SMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGN TLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKY YEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDV YLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKIN GELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDI LGNLYEVKSKKHPQIIKKGKRPAATKKAGQAKKKKGS" CDS 999..1019 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4200..4247 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4254..4403 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" polyA_signal 4432..4553 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_site 4438..4564 /label=SV40 Poly A Site promoter 4572..4812 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" repeat_region 4930..5070 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" rep_origin 5145..5600 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 5882..5986 /gene="bla" /label=AmpR promoter CDS 5987..6847 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 7018..7606 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 721 tctatataag cagagctctc tggctaacta ccggtgccac catggactac aaagaccatg 781 acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag cacggtccgc 841 gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc ctcgatgatt 901 ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac atgctcggca 961 gtgacgccct tgatgatttc gacctggaca tggttaaccc aaagaagaag cggaaggtcg 1021 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggcc atcggcatca 1081 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc 1141 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca 1201 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact 1261 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga 1321 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca 1381 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca 1441 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc 1501 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca 1561 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg 1621 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg 1681 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga 1741 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg 1801 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga 1861 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc 1921 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca 1981 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg 2041 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga 2101 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg 2161 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca 2221 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc 2281 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga 2341 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct 2401 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca 2461 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga 2521 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca 2581 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt 2641 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg 2701 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc 2761 tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac cccattccag tacctgagca 2821 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg 2881 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca 2941 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca 3001 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga 3061 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc 3121 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca 3181 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg 3241 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct 3301 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc 3361 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg 3421 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg 3481 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc 3541 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc 3601 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg 3661 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca 3721 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat 3781 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga 3841 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga 3901 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga 3961 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag 4021 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc 4081 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca 4141 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca 4201 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tccgacgcgc 4261 tggacgattt cgatctcgac atgctgggtt ctgatgccct cgatgacttt gacctggata 4321 tgttgggaag cgacgcattg gatgactttg atctggacat gctcggctcc gatgctctgg 4381 acgatttcga tctcgatatg ttataagaat tcatcgatac cgtcgagatc taacttgttt 4441 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4501 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 4561 tggaggtacc tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 4621 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 4681 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 4741 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 4801 tgtggaaagg acgaaacacc ggagaccacg gcaggtctca gttttagtac tctggaaaca 4861 gaatctacta aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt 4921 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 4981 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 5041 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 5101 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 5161 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 5221 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 5281 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 5341 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 5401 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 5461 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 5521 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 5581 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 5641 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 5701 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 5761 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 5821 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 5881 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 5941 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 6001 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 6061 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 6121 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 6181 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 6241 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 6301 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 6361 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 6421 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 6481 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 6541 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 6601 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 6661 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 6721 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 6781 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 6841 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 6901 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 6961 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 7021 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 7081 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 7141 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 7201 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 7261 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 7321 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 7381 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 7441 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 7501 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 7561 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 7621 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //