LOCUS Exported 8439 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses hAID-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-hAID-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8439) 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 8439) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8439 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 109..488 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 489..692 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 643..663 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 734..753 /label=T7 /note="T7 promoter, forward primer" promoter 734..752 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 760..769 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 766..1359 /codon_start=1 /gene="AICDA" /product="human activation-induced cytidine deaminase (Dickerson et al., 2003)" /label=AID /translation="MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLD FGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNL SLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWE GLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL" CDS 1408..5508 /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 5521..5769 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5782..5802 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5811..5828 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5851..5868) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5857..6081 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6152..6168) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6152..6168) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6165..6187) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6176..6192 /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(6200..6230) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6245..6266 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6383..6400) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6554..7142) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6634..6653) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7313..8173) /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 7936..7955 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8174..8278) /gene="bla" /label=AmpR promoter primer_bind complement(8357..8376) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 61 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 361 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 421 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 481 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 541 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 601 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 661 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctaga 721 gatccgcggc cgctaatacg actcactata gggagagccg ccaccatgga cagcctcttg 781 atgaaccgga ggaagtttct ttaccaattc aaaaatgtcc gctgggctaa gggtcggcgt 841 gagacctacc tgtgctacgt agtgaagagg cgtgacagtg ctacatcctt ttcactggac 901 tttggttatc ttcgcaataa gaacggctgc cacgtggaat tgctcttcct ccgctacatc 961 tcggactggg acctagaccc tggccgctgc taccgcgtca cctggttcac ctcctggagc 1021 ccctgctacg actgtgcccg acatgtggcc gactttctgc gagggaaccc caacctcagt 1081 ctgaggatct tcaccgcgcg cctctacttc tgtgaggacc gcaaggctga gcccgagggg 1141 ctgcggcggc tgcaccgcgc cggggtgcaa atagccatca tgaccttcaa agattatttt 1201 tactgctgga atacttttgt agaaaaccac gaaagaactt tcaaagcctg ggaagggctg 1261 catgaaaatt cagttcgtct ctccagacag cttcggcgca tccttttgcc cctgtatgag 1321 gttgatgact tacgagacgc atttcgtact ttgggactta gcggcagcga gactcccggg 1381 acctcagagt ccgccacacc cgaaagtgat aaaaagtatt ctattggttt agccatcggc 1441 actaattccg ttggatgggc tgtcataacc gatgaataca aagtaccttc aaagaaattt 1501 aaggtgttgg ggaacacaga ccgtcattcg attaaaaaga atcttatcgg tgccctccta 1561 ttcgatagtg gcgaaacggc agaggcgact cgcctgaaac gaaccgctcg gagaaggtat 1621 acacgtcgca agaaccgaat atgttactta caagaaattt ttagcaatga gatggccaaa 1681 gttgacgatt ctttctttca ccgtttggaa gagtccttcc ttgtcgaaga ggacaagaaa 1741 catgaacggc accccatctt tggaaacata gtagatgagg tggcatatca tgaaaagtac 1801 ccaacgattt atcacctcag aaaaaagcta gttgactcaa ctgataaagc ggacctgagg 1861 ttaatctact tggctcttgc ccatatgata aagttccgtg ggcactttct cattgagggt 1921 gatctaaatc cggacaactc ggatgtcgac aaactgttca tccagttagt acaaacctat 1981 aatcagttgt ttgaagagaa ccctataaat gcaagtggcg tggatgcgaa ggctattctt 2041 agcgcccgcc tctctaaatc ccgacggcta gaaaacctga tcgcacaatt acccggagag 2101 aagaaaaatg ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt 2161 aagtcgaact tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat 2221 gacgatctcg acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct 2281 gccaaaaacc ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt 2341 accaaggcgc cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg 2401 acacttctca aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt 2461 gatcagtcga aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc 2521 tacaagttta tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa 2581 ctcaatcgcg aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat 2641 caaatccact taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc 2701 ctcaaagaca atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg 2761 ggacccctgg cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg 2821 attactccat ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc 2881 gagaggatga ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt 2941 ttactttacg agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag 3001 ggcatgcgta aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta 3061 ttcaagacca accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt 3121 gaatgcttcg attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt 3181 acgtatcatg acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat 3241 gaagatatct tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt 3301 gaggaaagac taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag 3361 aggcgtcgct atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac 3421 aagcaaagtg gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac 3481 tttatgcagc tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag 3541 gtttccggac aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc 3601 atcaaaaagg gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga 3661 cgtcacaaac cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag 3721 gggcaaaaaa acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc 3781 agccagatct taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac 3841 ctctattacc tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt 3901 ttatctgatt acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc 3961 gacaataaag tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc 4021 gaggaagtcg taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata 4081 acgcaaagaa agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac 4141 aaggccggat ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca 4201 cagatactag attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa 4261 gtcaaagtaa tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc 4321 tataaagtta gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc 4381 gtagggaccg cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat 4441 tacaaagttt atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct 4501 acagccaaat acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg 4561 gcaaacggag agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc 4621 gtatgggata agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc 4681 aacatagtaa agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca 4741 aaaaggaata gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt 4801 ggcttcgata gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga 4861 aaatccaaga aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg 4921 tcttttgaaa agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag 4981 gatctcataa ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg 5041 atgttggcta gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac 5101 gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac 5161 gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa 5221 atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc 5281 gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg 5341 tttactctta ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat 5401 cgcaaacgat acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc 5461 acgggattat atgaaactcg gatagatttg tcacagcttg ggggtgactc tggtggttct 5521 actaatctgt cagatattat tgaaaaggag accggtaagc aactggttat ccaggaatcc 5581 atcctcatgc tcccagagga ggtggaagaa gtcattggga acaagccgga aagcgatata 5641 ctcgtgcaca ccgcctacga cgagagcacc gacgagaatg tcatgcttct gactagcgac 5701 gcccctgaat acaagccttg ggctctggtc atacaggata gcaacggtga gaacaagatt 5761 aagatgctct ctggtggttc tcccaagaag aagaggaaag tctaaccggt catcatcacc 5821 atcaccattg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat 5881 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 5941 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 6001 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 6061 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tcgataccgt 6121 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 6181 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg 6241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 6301 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 6361 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 6421 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 6481 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 6541 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 6601 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 6661 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 6721 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 6781 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 6841 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 6901 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 6961 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 7021 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7081 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7141 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7201 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 7261 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 7321 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 7381 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 7441 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 7501 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 7561 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 7621 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 7681 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 7741 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 7801 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 7861 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 7921 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 7981 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 8041 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 8101 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 8161 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 8221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 8281 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc 8341 tagggtcgac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 8401 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagca //