LOCUS       Exported                8415 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses hA3C-BE3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-hA3C-BE3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8415)
  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 8415)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8415
                     /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"
     CDS             1027..5127
                     /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             5140..5388
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5401..5421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5430..5447
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5470..5487)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5476..5700
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5771..5787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5771..5787)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5784..5806)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5795..5811
                     /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(5819..5849)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5864..5885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6002..6019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6173..6761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6253..6272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6932..7792)
                     /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     7555..7574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7793..7897)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7976..7995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8167..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 gaatccacag
      421 atcagaaacc cgatgaaggc aatgtatcca ggcacattct acttccaatt taaaaaccta
      481 tgggaagcca acgatcggaa cgaaacttgg ctgtgcttca ccgtggaagg tataaagcgc
      541 cgctcagttg tctcctggaa gacgggcgtc ttccgaaacc aggtggattc tgagacccat
      601 tgtcatgcag aaaggtgctt cctctcttgg ttctgcgacg acatactgtc tcctaacaca
      661 aagtaccagg tcacctggta cacatcttgg agcccttgcc cagactgtgc aggggaggtg
      721 gccgagttcc tggccaggca cagcaacgtg aatctcacca tcttcaccgc ccgcctctac
      781 tacttccagt atccatgtta ccaggagggg ctccgcagcc tgagtcagga aggggtcgct
      841 gtggagatca tggactatga agattttaaa tattgttggg aaaactttgt gtacaatgat
      901 aatgagccat tcaagccttg gaagggatta aaaaccaact ttcgacttct gaaaagaagg
      961 ctacgggaga gtctccagag cggcagcgag actcccggga cctcagagtc cgccacaccc
     1021 gaaagtgata aaaagtattc tattggttta gccatcggca ctaattccgt tggatgggct
     1081 gtcataaccg atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac
     1141 cgtcattcga ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca
     1201 gaggcgactc gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata
     1261 tgttacttac aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac
     1321 cgtttggaag agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt
     1381 ggaaacatag tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga
     1441 aaaaagctag ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc
     1501 catatgataa agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg
     1561 gatgtcgaca aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac
     1621 cctataaatg caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc
     1681 cgacggctag aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt
     1741 aaccttatag cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct
     1801 gaagatgcca aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg
     1861 gcacaaattg gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca
     1921 atcctcctat ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct
     1981 tcaatgatca aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc
     2041 cgtcagcaac tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac
     2101 gcaggttata ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata
     2161 ttagagaaga tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg
     2221 cgaaagcagc ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg
     2281 catgctatac ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag
     2341 attgagaaaa tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac
     2401 tctcggttcg catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag
     2461 gaagttgtcg ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac
     2521 aagaatttac cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca
     2581 gtgtacaatg aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt
     2641 ctaagcggag aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg
     2701 acagttaagc aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag
     2761 atctccgggg tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag
     2821 ataattaaag ataaggactt cctggataac gaagagaatg aagatatctt agaagatata
     2881 gtgttgactc ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac
     2941 gctcacctgt tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg
     3001 ggacgattgt cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt
     3061 ctcgattttc taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat
     3121 gactctttaa ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca
     3181 ttgcacgaac atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag
     3241 acagtcaaag tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt
     3301 gtaatcgaga tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag
     3361 cggatgaaga gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat
     3421 cctgtggaaa atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga
     3481 agggacatgt atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat
     3541 cacattgtac cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc
     3601 tcggataaga accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg
     3661 aagaactatt ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac
     3721 ttaactaaag ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt
     3781 cagctcgtgg aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg
     3841 aatacgaaat acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag
     3901 tcaaaattgg tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat
     3961 aactaccacc atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag
     4021 aaatacccga agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt
     4081 aagatgatcg cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat
     4141 tctaacatta tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa
     4201 cgacctttaa ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac
     4261 ttcgcgacgg tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag
     4321 gtgcagaccg gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc
     4381 atcgctcgta aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt
     4441 gcctattctg tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca
     4501 gtcaaagaat tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc
     4561 gacttccttg aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca
     4621 aagtatagtc tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag
     4681 cttcaaaagg ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg
     4741 tcccattacg agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt
     4801 gagcagcaca aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga
     4861 gtcatcctag ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat
     4921 aaacccatac gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc
     4981 gctccagccg cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc
     5041 aaggaggtgc tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg
     5101 atagatttgt cacagcttgg gggtgactct ggtggttcta ctaatctgtc agatattatt
     5161 gaaaaggaga ccggtaagca actggttatc caggaatcca tcctcatgct cccagaggag
     5221 gtggaagaag tcattgggaa caagccggaa agcgatatac tcgtgcacac cgcctacgac
     5281 gagagcaccg acgagaatgt catgcttctg actagcgacg cccctgaata caagccttgg
     5341 gctctggtca tacaggatag caacggtgag aacaagatta agatgctctc tggtggttct
     5401 cccaagaaga agaggaaagt ctaaccggtc atcatcacca tcaccattga gtttaaaccc
     5461 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5521 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5581 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5641 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     5701 cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg
     5761 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5821 acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca
     5881 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5941 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6001 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6061 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6121 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6181 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6241 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6301 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6361 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6421 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6481 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6541 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6601 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6661 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6721 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6781 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6841 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6901 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6961 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7021 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7081 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7141 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7201 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7261 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7321 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7381 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7441 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7501 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7561 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7621 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7681 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7741 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7801 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7861 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7921 ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa
     7981 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     8041 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     8101 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     8161 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     8221 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     8281 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     8341 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     8401 tacatcaagt gtatc
//
