LOCUS Exported 8454 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses hA3H-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-hA3H-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8454) 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 8454) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8454 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 403..412 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1066..5166 /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 5179..5427 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5440..5460 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5469..5486 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5509..5526) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5515..5739 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5810..5826) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5810..5826) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5823..5845) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5834..5850 /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(5858..5888) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5903..5924 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6041..6058) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6212..6800) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6292..6311) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6971..7831) /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 7594..7613 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7832..7936) /gene="bla" /label=AmpR promoter primer_bind complement(8015..8034) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 8206..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggctctgtta 421 acagccgaaa cattccgctt acagtttaac aacaagcgcc gcctcagaag gccttactac 481 ccgaggaagg ccctcttgtg ttaccagctg acgccgcaga atggctccac gcccacgaga 541 ggctactttg aaaacaagaa aaagtgccat gcagaaattt gctttattaa cgagatcaag 601 tccatgggac tggacgaaac gcagtgctac caagtcacct gttacctcac gtggagcccc 661 tgctcctcct gtgcctggga gctggttgac ttcatcaagg ctcacgacca tctgaacctg 721 ggcatcttcg cctcccgcct gtactaccac tggtgcaagc cccagcagaa ggggctgcgg 781 cttctgtgtg gatcccaggt cccggtggag gtcatgggct tcccagagtt tgctgactgc 841 tgggaaaact ttgtggacca cgagaaaccg ctttccttca acccctataa gatgttagag 901 gagctagata aaaacagtcg agccataaag cgacggcttg agaggataaa ggggctcctt 961 cttgttcttt tagattccag gggtacgtgc gcagggtcgt tacatggata tattgtgagc 1021 ggcagcgaga ctcccgggac ctcagagtcc gccacacccg aaagtgataa aaagtattct 1081 attggtttag ccatcggcac taattccgtt ggatgggctg tcataaccga tgaatacaaa 1141 gtaccttcaa agaaatttaa ggtgttgggg aacacagacc gtcattcgat taaaaagaat 1201 cttatcggtg ccctcctatt cgatagtggc gaaacggcag aggcgactcg cctgaaacga 1261 accgctcgga gaaggtatac acgtcgcaag aaccgaatat gttacttaca agaaattttt 1321 agcaatgaga tggccaaagt tgacgattct ttctttcacc gtttggaaga gtccttcctt 1381 gtcgaagagg acaagaaaca tgaacggcac cccatctttg gaaacatagt agatgaggtg 1441 gcatatcatg aaaagtaccc aacgatttat cacctcagaa aaaagctagt tgactcaact 1501 gataaagcgg acctgaggtt aatctacttg gctcttgccc atatgataaa gttccgtggg 1561 cactttctca ttgagggtga tctaaatccg gacaactcgg atgtcgacaa actgttcatc 1621 cagttagtac aaacctataa tcagttgttt gaagagaacc ctataaatgc aagtggcgtg 1681 gatgcgaagg ctattcttag cgcccgcctc tctaaatccc gacggctaga aaacctgatc 1741 gcacaattac ccggagagaa gaaaaatggg ttgttcggta accttatagc gctctcacta 1801 ggcctgacac caaattttaa gtcgaacttc gacttagctg aagatgccaa attgcagctt 1861 agtaaggaca cgtacgatga cgatctcgac aatctactgg cacaaattgg agatcagtat 1921 gcggacttat ttttggctgc caaaaacctt agcgatgcaa tcctcctatc tgacatactg 1981 agagttaata ctgagattac caaggcgccg ttatccgctt caatgatcaa aaggtacgat 2041 gaacatcacc aagacttgac acttctcaag gccctagtcc gtcagcaact gcctgagaaa 2101 tataaggaaa tattctttga tcagtcgaaa aacgggtacg caggttatat tgacggcgga 2161 gcgagtcaag aggaattcta caagtttatc aaacccatat tagagaagat ggatgggacg 2221 gaagagttgc ttgtaaaact caatcgcgaa gatctactgc gaaagcagcg gactttcgac 2281 aacggtagca ttccacatca aatccactta ggcgaattgc atgctatact tagaaggcag 2341 gaggattttt atccgttcct caaagacaat cgtgaaaaga ttgagaaaat cctaaccttt 2401 cgcatacctt actatgtggg acccctggcc cgagggaact ctcggttcgc atggatgaca 2461 agaaagtccg aagaaacgat tactccatgg aattttgagg aagttgtcga taaaggtgcg 2521 tcagctcaat cgttcatcga gaggatgacc aactttgaca agaatttacc gaacgaaaaa 2581 gtattgccta agcacagttt actttacgag tatttcacag tgtacaatga actcacgaaa 2641 gttaagtatg tcactgaggg catgcgtaaa cccgcctttc taagcggaga acagaagaaa 2701 gcaatagtag atctgttatt caagaccaac cgcaaagtga cagttaagca attgaaagag 2761 gactacttta agaaaattga atgcttcgat tctgtcgaga tctccggggt agaagatcga 2821 tttaatgcgt cacttggtac gtatcatgac ctcctaaaga taattaaaga taaggacttc 2881 ctggataacg aagagaatga agatatctta gaagatatag tgttgactct taccctcttt 2941 gaagatcggg aaatgattga ggaaagacta aaaacatacg ctcacctgtt cgacgataag 3001 gttatgaaac agttaaagag gcgtcgctat acgggctggg gacgattgtc gcggaaactt 3061 atcaacggga taagagacaa gcaaagtggt aaaactattc tcgattttct aaagagcgac 3121 ggcttcgcca ataggaactt tatgcagctg atccatgatg actctttaac cttcaaagag 3181 gatatacaaa aggcacaggt ttccggacaa ggggactcat tgcacgaaca tattgcgaat 3241 cttgctggtt cgccagccat caaaaagggc atactccaga cagtcaaagt agtggatgag 3301 ctagttaagg tcatgggacg tcacaaaccg gaaaacattg taatcgagat ggcacgcgaa 3361 aatcaaacga ctcagaaggg gcaaaaaaac agtcgagagc ggatgaagag aatagaagag 3421 ggtattaaag aactgggcag ccagatctta aaggagcatc ctgtggaaaa tacccaattg 3481 cagaacgaga aactttacct ctattaccta caaaatggaa gggacatgta tgttgatcag 3541 gaactggaca taaaccgttt atctgattac gacgtcgatc acattgtacc ccaatccttt 3601 ttgaaggacg attcaatcga caataaagtg cttacacgct cggataagaa ccgagggaaa 3661 agtgacaatg ttccaagcga ggaagtcgta aagaaaatga agaactattg gcggcagctc 3721 ctaaatgcga aactgataac gcaaagaaag ttcgataact taactaaagc tgagaggggt 3781 ggcttgtctg aacttgacaa ggccggattt attaaacgtc agctcgtgga aacccgccaa 3841 atcacaaagc atgttgcaca gatactagat tcccgaatga atacgaaata cgacgagaac 3901 gataagctga ttcgggaagt caaagtaatc actttaaagt caaaattggt gtcggacttc 3961 agaaaggatt ttcaattcta taaagttagg gagataaata actaccacca tgcgcacgac 4021 gcttatctta atgccgtcgt agggaccgca ctcattaaga aatacccgaa gctagaaagt 4081 gagtttgtgt atggtgatta caaagtttat gacgtccgta agatgatcgc gaaaagcgaa 4141 caggagatag gcaaggctac agccaaatac ttcttttatt ctaacattat gaatttcttt 4201 aagacggaaa tcactctggc aaacggagag atacgcaaac gacctttaat tgaaaccaat 4261 ggggagacag gtgaaatcgt atgggataag ggccgggact tcgcgacggt gagaaaagtt 4321 ttgtccatgc cccaagtcaa catagtaaag aaaactgagg tgcagaccgg agggttttca 4381 aaggaatcga ttcttccaaa aaggaatagt gataagctca tcgctcgtaa aaaggactgg 4441 gacccgaaaa agtacggtgg cttcgatagc cctacagttg cctattctgt cctagtagtg 4501 gcaaaagttg agaagggaaa atccaagaaa ctgaagtcag tcaaagaatt attggggata 4561 acgattatgg agcgctcgtc ttttgaaaag aaccccatcg acttccttga ggcgaaaggt 4621 tacaaggaag taaaaaagga tctcataatt aaactaccaa agtatagtct gtttgagtta 4681 gaaaatggcc gaaaacggat gttggctagc gccggagagc ttcaaaaggg gaacgaactc 4741 gcactaccgt ctaaatacgt gaatttcctg tatttagcgt cccattacga gaagttgaaa 4801 ggttcacctg aagataacga acagaagcaa ctttttgttg agcagcacaa acattatctc 4861 gacgaaatca tagagcaaat ttcggaattc agtaagagag tcatcctagc tgatgccaat 4921 ctggacaaag tattaagcgc atacaacaag cacagggata aacccatacg tgagcaggcg 4981 gaaaatatta tccatttgtt tactcttacc aacctcggcg ctccagccgc attcaagtat 5041 tttgacacaa cgatagatcg caaacgatac acttctacca aggaggtgct agacgcgaca 5101 ctgattcacc aatccatcac gggattatat gaaactcgga tagatttgtc acagcttggg 5161 ggtgactctg gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa 5221 ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 5281 aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 5341 atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 5401 aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc 5461 taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc 5521 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 5581 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 5641 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 5701 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 5761 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 5821 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 5881 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 5941 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 6001 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 6061 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 6121 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 6181 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 6241 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 6301 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 6361 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 6421 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 6481 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 6541 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 6601 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 6661 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6721 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6781 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6841 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 6901 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 6961 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 7021 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 7081 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 7141 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 7201 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 7261 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 7321 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 7381 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 7441 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 7501 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 7561 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 7621 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 7681 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 7741 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 7801 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 7861 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 7921 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 7981 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt 8041 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa 8101 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta 8161 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga 8221 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 8281 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 8341 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 8401 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatc //