LOCUS Exported 10177 bp ds-DNA circular SYN 01-MAR-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pnCas9-PBE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10177) AUTHORS . TITLE Direct Submission JOURNAL Exported Tuesday, May 30, 2017 from SnapGene 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10177 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 13..1999 /label=ubi promoter CDS 2021..2041 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 2042..2722 /label=rAPOBEC1 misc_feature 2723..2770 /label=XTEN misc_feature 2777..6925 /label=nCas9(D10A) CDS 6878..6925 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" misc_feature 6947..7207 /label=UGI CDS 7208..7228 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 7258..7981 /label=CaMV term polyA_signal 7302..7478 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" primer_bind complement(8021..8037) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 8045..8061 /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(8069..8099) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8114..8135 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(8423..9011) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9182..10042) /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" promoter complement(10043..10147) /gene="bla" /label=AmpR promoter ORIGIN 1 gagctcggta cctgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 61 gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 121 ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 181 atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 241 tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 301 tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 361 tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 421 gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 481 aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 541 actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 601 gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 661 ggcacggcat ctctgtcgct gcctctggac ccctctcgat cgagagttcc gctccaccgt 721 tggacttgct ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg 781 cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc 841 accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc 901 tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat 961 ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct 1021 accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca 1081 tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc 1141 gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc 1201 tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg 1261 catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg 1321 gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg 1381 ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta ttaattttgg 1441 atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata 1501 tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct 1561 ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat 1621 cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt 1681 gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg 1741 tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt 1801 catatgctct aaccttgagt acctatctat tataataaac aagtatgttt tataattatt 1861 ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc 1921 ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt 1981 gtttggtgtt acttctgcaa agcttgtcga cggatccatg ccaaagaaga agaggaaggt 2041 ttcatcggag accggccctg ttgctgttga ccccaccctg cggcggagaa tcgagccaca 2101 cgagttcgag gtgttcttcg acccaaggga gctccgcaag gagacgtgcc tcctgtacga 2161 gatcaactgg ggcggcaggc actccatctg gaggcacacc agccaaaaca ccaacaagca 2221 cgtggaggtc aacttcatcg agaagttcac caccgagagg tacttctgcc caaacacccg 2281 ctgctccatc acctggttcc tgtcctggag cccatgcggc gagtgctcca gggccatcac 2341 cgagttcctc agccgctacc cacacgtcac cctgttcatc tacatcgcca ggctctacca 2401 ccacgccgac ccaaggaaca ggcagggcct ccgcgacctg atctccagcg gcgtgaccat 2461 ccaaatcatg accgagcagg agtccggcta ctgctggagg aacttcgtca actactcccc 2521 aagcaacgag gcccactggc caaggtaccc acacctctgg gtgcgcctct acgtgctcga 2581 gctgtactgc atcatcctcg gcctgccacc atgcctcaac atcctgaggc gcaagcaacc 2641 acagctgacc ttcttcacca tcgccctcca aagctgccac taccagaggc tcccaccaca 2701 catcctgtgg gctaccggcc tcaagtccgg cagcgagacg ccaggcacct ccgagagcgc 2761 tacgcctgaa cttaaggaca agaagtactc gatcggcctc gccatcggga cgaactcagt 2821 tggctgggcc gtgatcaccg acgagtacaa ggtgccctct aagaagttca aggtcctggg 2881 gaacaccgac cgccattcca tcaagaagaa cctcatcggc gctctcctgt tcgacagcgg 2941 ggagaccgct gaggctacga ggctcaagag aaccgctagg cgccggtaca cgagaaggaa 3001 gaacaggatc tgctacctcc aagagatttt ctccaacgag atggccaagg ttgacgattc 3061 attcttccac cgcctggagg agtctttcct cgtggaggag gataagaagc acgagcggca 3121 tcccatcttc ggcaacatcg tggacgaggt tgcctaccac gagaagtacc ctacgatcta 3181 ccatctgcgg aagaagctcg tggactccac cgataaggcg gacctcagac tgatctacct 3241 cgctctggcc cacatgatca agttccgcgg ccatttcctg atcgaggggg atctcaaccc 3301 agacaacagc gatgttgaca agctgttcat ccaactcgtg cagacctaca accaactctt 3361 cgaggagaac ccgatcaacg cctctggcgt ggacgcgaag gctatcctgt ccgcgaggct 3421 ctcgaagtcc aggaggctgg agaacctgat cgctcagctc ccaggcgaga agaagaacgg 3481 cctgttcggg aacctcatcg ctctcagcct ggggctcacc ccgaacttca agtcgaactt 3541 cgatctcgct gaggacgcca agctgcaact ctccaaggac acctacgacg atgacctcga 3601 taacctcctg gcccagatcg gcgatcaata cgcggacctg ttcctcgctg ccaagaacct 3661 gtcggacgcc atcctcctgt cagatatcct ccgcgtgaac accgagatca cgaaggctcc 3721 actctctgcc tccatgatca agcgctacga cgagcaccat caggatctga ccctcctgaa 3781 ggcgctggtc cgccaacagc tcccggagaa gtacaaggag attttcttcg atcagtcgaa 3841 gaacggctac gctgggtaca tcgacggcgg ggcctcacaa gaggagttct acaagttcat 3901 caagccaatc ctggagaaga tggacggcac ggaggagctc ctggtgaagc tcaacaggga 3961 ggacctcctg cggaagcaga gaaccttcga taacggcagc atcccccacc aaatccatct 4021 cggggagctg cacgccatcc tgagaaggca agaggacttc taccctttcc tcaaggataa 4081 ccgggagaag atcgagaaga tcctgacctt cagaatccca tactacgtcg gccctctcgc 4141 gcgggggaac tcaagattcg cttggatgac ccgcaagtct gaggagacca tcacgccgtg 4201 gaacttcgag gaggtggtgg acaagggcgc tagcgctcag tcgttcatcg agaggatgac 4261 caacttcgac aagaacctgc ccaacgagaa ggtgctccct aagcactcgc tcctgtacga 4321 gtacttcacc gtctacaacg agctcacgaa ggtgaagtac gtcaccgagg gcatgcgcaa 4381 gccagcgttc ctgtccgggg agcagaagaa ggctatcgtg gacctcctgt tcaagaccaa 4441 ccggaaggtc acggttaagc aactcaagga ggactacttc aagaagatcg agtgcttcga 4501 ttcggtcgag atcagcggcg ttgaggaccg cttcaacgcc agcctcggga cctaccacga 4561 tctcctgaag atcatcaagg ataaggactt cctggacaac gaggagaacg aggatatcct 4621 ggaggacatc gtgctgaccc tcacgctgtt cgaggacagg gagatgatcg aggagcgcct 4681 gaagacgtac gcccatctct tcgatgacaa ggtcatgaag caactcaagc gccggagata 4741 caccggctgg gggaggctgt cccgcaagct catcaacggc atccgggaca agcagtccgg 4801 gaagaccatc ctcgacttcc tcaagagcga tggcttcgcc aacaggaact tcatgcaact 4861 gatccacgat gacagcctca ccttcaagga ggatatccaa aaggctcaag tgagcggcca 4921 gggggactcg ctgcacgagc atatcgcgaa cctcgctggc tcccccgcga tcaagaaggg 4981 catcctccag accgtgaagg ttgtggacga gctcgtgaag gtcatgggcc ggcacaagcc 5041 tgagaacatc gtcatcgaga tggccagaga gaaccaaacc acgcagaagg ggcaaaagaa 5101 ctctagggag cgcatgaagc gcatcgagga gggcatcaag gagctggggt cccaaatcct 5161 caaggagcac ccagtggaga acacccaact gcagaacgag aagctctacc tgtactacct 5221 ccagaacggc agggatatgt acgtggacca agagctggat atcaaccgcc tcagcgatta 5281 cgacgtcgat catatcgttc cccagtcttt cctgaaggat gactccatcg acaacaaggt 5341 cctcaccagg tcggacaaga accgcggcaa gtcagataac gttccatctg aggaggtcgt 5401 taagaagatg aagaactact ggaggcagct cctgaacgcc aagctgatca cgcaaaggaa 5461 gttcgacaac ctcaccaagg ctgagagagg cgggctctca gagctggaca aggccggctt 5521 catcaagcgg cagctggtcg agaccagaca aatcacgaag cacgttgcgc aaatcctcga 5581 ctctcggatg aacacgaagt acgatgagaa cgacaagctg atcagggagg ttaaggtgat 5641 caccctgaag tctaagctcg tctccgactt caggaaggat ttccagttct acaaggttcg 5701 cgagatcaac aactaccacc atgcccatga cgcttacctc aacgctgtgg tcggcaccgc 5761 tctgatcaag aagtacccaa agctggagtc cgagttcgtg tacggggact acaaggttta 5821 cgatgtgcgc aagatgatcg ccaagtcgga gcaagagatc ggcaaggcta ccgccaagta 5881 cttcttctac tcaaacatca tgaacttctt caagaccgag atcacgctgg ccaacggcga 5941 gatccggaag agaccgctca tcgagaccaa cggcgagacg ggggagatcg tgtgggacaa 6001 gggcagggat ttcgcgaccg tccgcaaggt tctctccatg ccccaggtga acatcgtcaa 6061 gaagaccgag gtccaaacgg gcgggttctc aaaggagtct atcctgccta agcggaacag 6121 cgacaagctc atcgccagaa agaaggactg ggacccaaag aagtacggcg ggttcgacag 6181 ccctaccgtg gcctactcgg tcctggttgt ggcgaaggtt gagaagggca agtccaagaa 6241 gctcaagagc gtgaaggagc tcctggggat caccatcatg gagaggtcca gcttcgagaa 6301 gaacccaatc gacttcctgg aggccaaggg ctacaaggag gtgaagaagg acctgatcat 6361 caagctcccg aagtactctc tcttcgagct ggagaacggc aggaagagaa tgctggcttc 6421 cgctggcgag ctccagaagg ggaacgagct cgcgctgcca agcaagtacg tgaacttcct 6481 ctacctggct tcccactacg agaagctcaa gggcagcccg gaggacaacg agcaaaagca 6541 gctgttcgtc gagcagcaca agcattacct cgacgagatc atcgagcaaa tctccgagtt 6601 cagcaagcgc gtgatcctcg ccgacgcgaa cctggataag gtcctctccg cctacaacaa 6661 gcaccgggac aagcccatca gagagcaagc ggagaacatc atccatctct tcaccctgac 6721 gaacctcggc gctcctgctg ctttcaagta cttcgacacc acgatcgatc ggaagagata 6781 cacctccacg aaggaggtcc tggacgcgac cctcatccac cagtcgatca ccggcctgta 6841 cgagacgagg atcgacctct cacaactcgg cggggataag agacccgcag caaccaagaa 6901 ggcagggcaa gcaaagaaga agaagacgcg tgactccggc ggcagcacca acctgtccga 6961 catcatcgag aaggagacgg gcaagcaact cgtgatccag gagagcatcc tcatgctgcc 7021 agaggaggtg gaggaggtca tcggcaacaa gccagagtcc gacatcctgg tgcacaccgc 7081 ctacgacgag tccaccgacg agaacgtcat gctcctgacc agcgacgccc cagagtacaa 7141 gccatgggcc ctcgtcatcc aggacagcaa cggggagaac aagatcaaga tgctgtcggg 7201 ggggagccca aagaagaagc ggaaggtgta gtgtacaagt aacccgggaa ttcggtacgc 7261 tgaaatcacc agtctctctc tacaaatcta tctctctcta ttttctccat aaataatgtg 7321 tgagtagttt cccgataagg gaaattaggg ttcttatagg gtttcgctca tgtgttgagc 7381 atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct 7441 aattcctaaa accaaaatcc agtactaaaa tccagatctc ctaaagtccc tatagatctt 7501 tgtcgtgaat ataaaccaga cacgagacga ctaaacctgg agcccagacg ccgttcgaag 7561 ctagaagtac cgcttaggca ggaggccgtt agggaaaaga tgctaaggca gggttggtta 7621 cgttgactcc cccgtaggtt tggtttaaat atgatgaagt ggacggaagg aaggaggaag 7681 acaaggaagg ataaggttgc aggccctgtg caaggtaaga agatggaaat ttgatagagg 7741 tacgctacta tacttatact atacgctaag ggaatgcttg tatttatacc ctataccccc 7801 taataacccc ttatcaattt aagaaataat ccgcataagc ccccgcttaa aaattggtat 7861 cagagccatg aataggtcta tgaccaaaac tcaagaggat aaaacctcac caaaatacga 7921 aagagttctt aactctaaag ataaaagatc tttcaagatc aaaactagtt ccctcacacc 7981 ggagcatgcg atatcctcga gagatctagg cgtaatcatg gtcatagctg tttcctgtgt 8041 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 8101 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 8161 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 8221 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 8281 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 8341 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 8401 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 8461 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 8521 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 8581 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 8641 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 8701 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 8761 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 8821 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 8881 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 8941 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 9001 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 9061 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 9121 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 9181 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 9241 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 9301 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 9361 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 9421 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 9481 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 9541 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 9601 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 9661 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 9721 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 9781 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 9841 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 9901 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 9961 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 10021 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 10081 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 10141 ttccgcgcac atttccccga aaagtgccac ctgacgt //