LOCUS Exported 8964 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses hA3F-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-hA3F-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8964) AUTHORS Wang X, Li J, Wang Y, Yang V, Wei J, Wu J, Wang R, Huang X, Chen J, Yang L TITLE Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. JOURNAL Nat Biotechnol., 2018 REFERENCE 2 (bases 1 to 8964) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8964 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 96..475 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 476..679 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 630..650 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 721..740 /label=T7 /note="T7 promoter, forward primer" promoter 721..739 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1920..6020 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6033..6281 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 6294..6314 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6323..6340 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6363..6380) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6369..6593 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6664..6680) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6664..6680) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6677..6699) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6688..6704 /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(6712..6742) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6757..6778 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6895..6912) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7066..7654) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7146..7165) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7825..8685) /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" primer_bind 8448..8467 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8686..8790) /gene="bla" /label=AmpR promoter primer_bind complement(8869..8888) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc 61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 661 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc 721 taatacgact cactataggg agagccgcca ccatgaagcc tcacttcaga aacacagtgg 781 agcgaatgta tcgagacaca ttctcctaca acttttataa tagacccatc ctttctcgtc 841 ggaataccgt ctggctgtgc tacgaagtga aaacaaaggg tccctcaagg ccccgtttgg 901 acgcaaagat ctttcgaggc caggtgtatt cccagcctga gcaccacgca gaaatgtgct 961 tcctctcttg gttctgtggc aaccagctgc ctgcttacaa gtgtttccag atcacctggt 1021 ttgtatcctg gaccccctgc ccggactgtg tggcgaagct ggccgaattc ctggctgagc 1081 accccaatgt caccctgacc atctccgccg cccgcctcta ctactactgg gaaagagatt 1141 accgaagggc gctctgcagg ctgagtcagg caggggcccg cgtgaagatt atggacgatg 1201 aagaatttgc atactgctgg gaaaactttg tgtacagtga aggtcagcca ttcatgcctt 1261 ggtacaaatt cgatgacaat tatgcattcc tgcaccgcac gctaaaggag attctcagaa 1321 acccgatgga ggcaatgtat ccacacatat tctacttcca ctttaaaaac ctacgcaaag 1381 cctatggtcg gaacgaaagc tggctgtgct tcaccatgga agttgtaaag caccactcac 1441 ctgtctcctg gaagaggggc gtcttccgaa accaggtgga tcctgagacc cattgtcatg 1501 cagaaaggtg cttcctctct tggttctgtg acgacatact gtctcctaac acaaactacg 1561 aggtcacctg gtacacatct tggagccctt gcccagagtg tgcaggggag gtggccgagt 1621 tcctggccag gcacagcaac gtgaatctca ccatcttcac cgcccgcctc tactacttct 1681 gggatacaga ttaccaggag gggctccgca gcctgagtca ggaaggggcc tccgtggaga 1741 tcatgggcta caaagatttt aaatattgtt gggaaaactt tgtgtacaat gatgatgagc 1801 cattcaagcc ttggaaagga ctaaaataca actttctatt cctggacagc aagctgcagg 1861 agattctcga gagcggcagc gagactcccg ggacctcaga gtccgccaca cccgaaagtg 1921 ataaaaagta ttctattggt ttagccatcg gcactaattc cgttggatgg gctgtcataa 1981 ccgatgaata caaagtacct tcaaagaaat ttaaggtgtt ggggaacaca gaccgtcatt 2041 cgattaaaaa gaatcttatc ggtgccctcc tattcgatag tggcgaaacg gcagaggcga 2101 ctcgcctgaa acgaaccgct cggagaaggt atacacgtcg caagaaccga atatgttact 2161 tacaagaaat ttttagcaat gagatggcca aagttgacga ttctttcttt caccgtttgg 2221 aagagtcctt ccttgtcgaa gaggacaaga aacatgaacg gcaccccatc tttggaaaca 2281 tagtagatga ggtggcatat catgaaaagt acccaacgat ttatcacctc agaaaaaagc 2341 tagttgactc aactgataaa gcggacctga ggttaatcta cttggctctt gcccatatga 2401 taaagttccg tgggcacttt ctcattgagg gtgatctaaa tccggacaac tcggatgtcg 2461 acaaactgtt catccagtta gtacaaacct ataatcagtt gtttgaagag aaccctataa 2521 atgcaagtgg cgtggatgcg aaggctattc ttagcgcccg cctctctaaa tcccgacggc 2581 tagaaaacct gatcgcacaa ttacccggag agaagaaaaa tgggttgttc ggtaacctta 2641 tagcgctctc actaggcctg acaccaaatt ttaagtcgaa cttcgactta gctgaagatg 2701 ccaaattgca gcttagtaag gacacgtacg atgacgatct cgacaatcta ctggcacaaa 2761 ttggagatca gtatgcggac ttatttttgg ctgccaaaaa ccttagcgat gcaatcctcc 2821 tatctgacat actgagagtt aatactgaga ttaccaaggc gccgttatcc gcttcaatga 2881 tcaaaaggta cgatgaacat caccaagact tgacacttct caaggcccta gtccgtcagc 2941 aactgcctga gaaatataag gaaatattct ttgatcagtc gaaaaacggg tacgcaggtt 3001 atattgacgg cggagcgagt caagaggaat tctacaagtt tatcaaaccc atattagaga 3061 agatggatgg gacggaagag ttgcttgtaa aactcaatcg cgaagatcta ctgcgaaagc 3121 agcggacttt cgacaacggt agcattccac atcaaatcca cttaggcgaa ttgcatgcta 3181 tacttagaag gcaggaggat ttttatccgt tcctcaaaga caatcgtgaa aagattgaga 3241 aaatcctaac ctttcgcata ccttactatg tgggacccct ggcccgaggg aactctcggt 3301 tcgcatggat gacaagaaag tccgaagaaa cgattactcc atggaatttt gaggaagttg 3361 tcgataaagg tgcgtcagct caatcgttca tcgagaggat gaccaacttt gacaagaatt 3421 taccgaacga aaaagtattg cctaagcaca gtttacttta cgagtatttc acagtgtaca 3481 atgaactcac gaaagttaag tatgtcactg agggcatgcg taaacccgcc tttctaagcg 3541 gagaacagaa gaaagcaata gtagatctgt tattcaagac caaccgcaaa gtgacagtta 3601 agcaattgaa agaggactac tttaagaaaa ttgaatgctt cgattctgtc gagatctccg 3661 gggtagaaga tcgatttaat gcgtcacttg gtacgtatca tgacctccta aagataatta 3721 aagataagga cttcctggat aacgaagaga atgaagatat cttagaagat atagtgttga 3781 ctcttaccct ctttgaagat cgggaaatga ttgaggaaag actaaaaaca tacgctcacc 3841 tgttcgacga taaggttatg aaacagttaa agaggcgtcg ctatacgggc tggggacgat 3901 tgtcgcggaa acttatcaac gggataagag acaagcaaag tggtaaaact attctcgatt 3961 ttctaaagag cgacggcttc gccaatagga actttatgca gctgatccat gatgactctt 4021 taaccttcaa agaggatata caaaaggcac aggtttccgg acaaggggac tcattgcacg 4081 aacatattgc gaatcttgct ggttcgccag ccatcaaaaa gggcatactc cagacagtca 4141 aagtagtgga tgagctagtt aaggtcatgg gacgtcacaa accggaaaac attgtaatcg 4201 agatggcacg cgaaaatcaa acgactcaga aggggcaaaa aaacagtcga gagcggatga 4261 agagaataga agagggtatt aaagaactgg gcagccagat cttaaaggag catcctgtgg 4321 aaaataccca attgcagaac gagaaacttt acctctatta cctacaaaat ggaagggaca 4381 tgtatgttga tcaggaactg gacataaacc gtttatctga ttacgacgtc gatcacattg 4441 taccccaatc ctttttgaag gacgattcaa tcgacaataa agtgcttaca cgctcggata 4501 agaaccgagg gaaaagtgac aatgttccaa gcgaggaagt cgtaaagaaa atgaagaact 4561 attggcggca gctcctaaat gcgaaactga taacgcaaag aaagttcgat aacttaacta 4621 aagctgagag gggtggcttg tctgaacttg acaaggccgg atttattaaa cgtcagctcg 4681 tggaaacccg ccaaatcaca aagcatgttg cacagatact agattcccga atgaatacga 4741 aatacgacga gaacgataag ctgattcggg aagtcaaagt aatcacttta aagtcaaaat 4801 tggtgtcgga cttcagaaag gattttcaat tctataaagt tagggagata aataactacc 4861 accatgcgca cgacgcttat cttaatgccg tcgtagggac cgcactcatt aagaaatacc 4921 cgaagctaga aagtgagttt gtgtatggtg attacaaagt ttatgacgtc cgtaagatga 4981 tcgcgaaaag cgaacaggag ataggcaagg ctacagccaa atacttcttt tattctaaca 5041 ttatgaattt ctttaagacg gaaatcactc tggcaaacgg agagatacgc aaacgacctt 5101 taattgaaac caatggggag acaggtgaaa tcgtatggga taagggccgg gacttcgcga 5161 cggtgagaaa agttttgtcc atgccccaag tcaacatagt aaagaaaact gaggtgcaga 5221 ccggagggtt ttcaaaggaa tcgattcttc caaaaaggaa tagtgataag ctcatcgctc 5281 gtaaaaagga ctgggacccg aaaaagtacg gtggcttcga tagccctaca gttgcctatt 5341 ctgtcctagt agtggcaaaa gttgagaagg gaaaatccaa gaaactgaag tcagtcaaag 5401 aattattggg gataacgatt atggagcgct cgtcttttga aaagaacccc atcgacttcc 5461 ttgaggcgaa aggttacaag gaagtaaaaa aggatctcat aattaaacta ccaaagtata 5521 gtctgtttga gttagaaaat ggccgaaaac ggatgttggc tagcgccgga gagcttcaaa 5581 aggggaacga actcgcacta ccgtctaaat acgtgaattt cctgtattta gcgtcccatt 5641 acgagaagtt gaaaggttca cctgaagata acgaacagaa gcaacttttt gttgagcagc 5701 acaaacatta tctcgacgaa atcatagagc aaatttcgga attcagtaag agagtcatcc 5761 tagctgatgc caatctggac aaagtattaa gcgcatacaa caagcacagg gataaaccca 5821 tacgtgagca ggcggaaaat attatccatt tgtttactct taccaacctc ggcgctccag 5881 ccgcattcaa gtattttgac acaacgatag atcgcaaacg atacacttct accaaggagg 5941 tgctagacgc gacactgatt caccaatcca tcacgggatt atatgaaact cggatagatt 6001 tgtcacagct tgggggtgac tctggtggtt ctactaatct gtcagatatt attgaaaagg 6061 agaccggtaa gcaactggtt atccaggaat ccatcctcat gctcccagag gaggtggaag 6121 aagtcattgg gaacaagccg gaaagcgata tactcgtgca caccgcctac gacgagagca 6181 ccgacgagaa tgtcatgctt ctgactagcg acgcccctga atacaagcct tgggctctgg 6241 tcatacagga tagcaacggt gagaacaaga ttaagatgct ctctggtggt tctcccaaga 6301 agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc 6361 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 6421 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 6481 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 6541 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 6601 ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc 6661 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 6721 agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat 6781 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 6841 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 6901 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 6961 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 7021 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 7081 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 7141 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 7201 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 7261 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 7321 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 7381 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 7441 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 7501 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 7561 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 7621 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 7681 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 7741 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 7801 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 7861 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7921 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 7981 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 8041 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 8101 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 8161 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 8221 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 8281 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 8341 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 8401 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 8461 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 8521 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 8581 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 8641 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 8701 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 8761 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 8821 cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc 8881 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta 8941 gtgcgcgagc aaaatttaag ctac //