LOCUS Exported 7930 bp ds-DNA circular SYN 22-NOV-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 7930) AUTHORS Trial User TITLE Direct Submission JOURNAL Exported Nov 23, 2018 from SnapGene Viewer 4.2.6 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7930 /organism="recombinant plasmid" /mol_type="other DNA" misc_feature complement(19..102) /label=SV40_enhancer /note="SV40_enhancer" protein_bind 36..46 /label=NFkB2 /note="NFkB2" misc_feature 126..867 /label=CMV IE /note="CMV IE" protein_bind 132..145 /label=NF1 /note="NF1" protein_bind 179..192 /label=NF1 /note="NF1" protein_bind 233..246 /label=NF1 /note="NF1" enhancer 263..642 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 288..840 /label=CMV_immearly_promoter misc_feature 343..630 /label=CAG_enhancer promoter 643..846 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 786..867 /label=CMV_promoter misc_feature 797..817 /label=CMV_fwd_primer TATA_signal 832..837 /label="" /note="" intron 938..1070 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" misc_feature 938..1070 /label=chim_int gene 1106..3259 /label=hCas9 /note="hCas9" misc_feature 1109..1261 /label=C-intein /note="C-intein" misc_feature 1262..1273 /label=Disordered /note="Disordered" CDS 1710..1721 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" misc_feature 3227..3259 /label=NLS with linker /note="NLS with linker" CDS 3239..3259 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" sig_peptide 3239..3259 /label=SV40 NLS /note="SV40 NLS" 3'UTR 3270..3471 /label=SV40 pA /note="SV40 pA" misc_feature complement(3270..3289) /label=EBV_rev_primer polyA_signal 3299..3380 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 3369..3374 /label="" /note="" enhancer 3478..3857 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3858..4061 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 4106..4124 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 4144..4407 /codon_start=1 /label=AcrIIA4 /translation="MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISE SENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN" misc_feature 4447..4468 /label=mir122 binding site misc_feature 4476..4497 /label=mir122 binding site polyA_signal 4530..4754 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4808..4937 /label=ITR /note="dnas_title=ITR vntifkey=34" primer_bind complement(4975..4991) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 4999..5015 /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(5023..5053) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5068..5089 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5377..5965) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6136..6996) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6997..7101) /gene="bla" /label=AmpR promoter rep_origin complement(7128..7583) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 7725..7741 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 7760..7889 /label=ITR /note="dnas_title=ITR vntifkey=34" ORIGIN 1 ggccgccatt atggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac cagtcccggt ctcctcccat 121 gcatgtcaat attggccatt agccatatta ttcattggtt atatagcata aatcaatatt 181 ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta tattggctca 241 tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt 301 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 361 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 421 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 481 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc 541 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct 601 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 661 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 721 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat 781 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 841 agaggtcgtt tagtgaaccg tcagatcact agtagcttta ttgcggtagt ttatcacagt 901 taaattgcta acgcagtcag tgctcgactg atcacaggta agtatcaagg ttacaagaca 961 ggtttaagga ggccaataga aactgggctt gtcgagacag agaagattct tgcgtttctg 1021 ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag gggtaccgaa 1081 gccgctagcg ctaccggtcg ccaccatggc ggcggcgtgc ccggaactgc gtcagctggc 1141 gcagagcgat gtgtattggg atccgattgt gagcattgaa ccggatggcg tggaagaagt 1201 gtttgatctg accgtgccgg gcccgcataa ctttgtggcg aacgatatta ttgcgcataa 1261 ctctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat 1321 caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag 1381 gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg 1441 acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc 1501 ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct 1561 gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct 1621 ctccgactac gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga 1681 taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga 1741 agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac 1801 acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa 1861 agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca 1921 aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt 1981 gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta 2041 taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt 2101 aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta 2161 taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac 2221 cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc 2281 caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt 2341 gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa 2401 catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa 2461 aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg 2521 attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa 2581 gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag 2641 cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga 2701 cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat 2761 gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt 2821 taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga 2881 gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat 2941 aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc 3001 ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt 3061 tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag 3121 aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac 3181 ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc 3241 caagaagaag aggaaggtgt ctcgagctcg atgagtttgg acaaaccaca actagaatgc 3301 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta 3361 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg 3421 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atctagagac 3481 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 3541 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 3601 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 3661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 3721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 3781 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 3841 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 3901 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 3961 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 4021 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac 4081 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct 4141 agcatgaaca ttaacgacct gattcgggaa ataaagaaca aggactacac ggtcaagctg 4201 tcaggcaccg actccaactc tataacacag ctcattataa gggttaataa tgatggcaac 4261 gaatatgtca tctctgaatc cgagaatgaa tcaatcgtgg agaaattcat cagtgcattt 4321 aaaaacggct ggaaccagga gtacgaagat gaggaagagt tctacaatga tatgcaaact 4381 atcacactga aatctgagct gaactagctc gagcggccgc cactgtgctg gatatctgca 4441 gaattacaaa caccattgtc acactccatt atatacaaac accattgtca cactccatag 4501 cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 4561 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 4621 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 4681 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 4741 ggtgggctct atggacgcgt gcggccgcag gtagataagt agcatggcgg gttaatcatt 4801 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 4861 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 4921 agcgagcgag cgcgcagcct aattaaggcc ttaattaaat ctggcgtaat catggtcata 4981 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5041 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5101 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5161 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaac cgcagcaaaa 5341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 5521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 5581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 5641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 5701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 5761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 5821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 5881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 5941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 6001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 6061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 6121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 6181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 6241 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 6301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 6361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 6421 taatagtttg cgcaacgttg ttaccattac tacaggcatc gtggtgtcac gctcgtcgtt 6481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 6541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 6601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 6661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 6721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 6781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 6841 accactattg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 6901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 6961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 7021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 7081 taaacaaata ggggttccgc gcacatttcc ccgaaagatg ccacctgaaa ttataaacgt 7141 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 7201 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 7261 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 7321 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 7381 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 7441 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 7501 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 7561 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg 7621 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 7681 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 7741 tgaattaggt taattaaggc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 7801 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 7861 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt 7921 atctacctgc //