pCMV-hA3C-BE3

LOCUS       pCMV-hA3C-BE3                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.biofeng.com
FEATURES             Location/Qualifiers
     source          1..8415
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        76..455
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        456..659
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     610..630
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     701..720
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        701..719
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1351..5451
                     /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             5464..5712
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5725..5745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5754..5771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5794..5811)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5800..6024
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6095..6111)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6095..6111)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6108..6130)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6119..6135
                     /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(6143..6173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6188..6209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6326..6343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6497..7085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6577..6596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7256..8116)
                     /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     7879..7898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8117..8221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8300..8319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
       61 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
      121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      301 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      361 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      421 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      481 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      541 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      601 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      661 gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc taatacgact cactataggg
      721 agagccgcca ccatgaatcc acagatcaga aacccgatga aggcaatgta tccaggcaca
      781 ttctacttcc aatttaaaaa cctatgggaa gccaacgatc ggaacgaaac ttggctgtgc
      841 ttcaccgtgg aaggtataaa gcgccgctca gttgtctcct ggaagacggg cgtcttccga
      901 aaccaggtgg attctgagac ccattgtcat gcagaaaggt gcttcctctc ttggttctgc
      961 gacgacatac tgtctcctaa cacaaagtac caggtcacct ggtacacatc ttggagccct
     1021 tgcccagact gtgcagggga ggtggccgag ttcctggcca ggcacagcaa cgtgaatctc
     1081 accatcttca ccgcccgcct ctactacttc cagtatccat gttaccagga ggggctccgc
     1141 agcctgagtc aggaaggggt cgctgtggag atcatggact atgaagattt taaatattgt
     1201 tgggaaaact ttgtgtacaa tgataatgag ccattcaagc cttggaaggg attaaaaacc
     1261 aactttcgac ttctgaaaag aaggctacgg gagagtctcc agagcggcag cgagactccc
     1321 gggacctcag agtccgccac acccgaaagt gataaaaagt attctattgg tttagccatc
     1381 ggcactaatt ccgttggatg ggctgtcata accgatgaat acaaagtacc ttcaaagaaa
     1441 tttaaggtgt tggggaacac agaccgtcat tcgattaaaa agaatcttat cggtgccctc
     1501 ctattcgata gtggcgaaac ggcagaggcg actcgcctga aacgaaccgc tcggagaagg
     1561 tatacacgtc gcaagaaccg aatatgttac ttacaagaaa tttttagcaa tgagatggcc
     1621 aaagttgacg attctttctt tcaccgtttg gaagagtcct tccttgtcga agaggacaag
     1681 aaacatgaac ggcaccccat ctttggaaac atagtagatg aggtggcata tcatgaaaag
     1741 tacccaacga tttatcacct cagaaaaaag ctagttgact caactgataa agcggacctg
     1801 aggttaatct acttggctct tgcccatatg ataaagttcc gtgggcactt tctcattgag
     1861 ggtgatctaa atccggacaa ctcggatgtc gacaaactgt tcatccagtt agtacaaacc
     1921 tataatcagt tgtttgaaga gaaccctata aatgcaagtg gcgtggatgc gaaggctatt
     1981 cttagcgccc gcctctctaa atcccgacgg ctagaaaacc tgatcgcaca attacccgga
     2041 gagaagaaaa atgggttgtt cggtaacctt atagcgctct cactaggcct gacaccaaat
     2101 tttaagtcga acttcgactt agctgaagat gccaaattgc agcttagtaa ggacacgtac
     2161 gatgacgatc tcgacaatct actggcacaa attggagatc agtatgcgga cttatttttg
     2221 gctgccaaaa accttagcga tgcaatcctc ctatctgaca tactgagagt taatactgag
     2281 attaccaagg cgccgttatc cgcttcaatg atcaaaaggt acgatgaaca tcaccaagac
     2341 ttgacacttc tcaaggccct agtccgtcag caactgcctg agaaatataa ggaaatattc
     2401 tttgatcagt cgaaaaacgg gtacgcaggt tatattgacg gcggagcgag tcaagaggaa
     2461 ttctacaagt ttatcaaacc catattagag aagatggatg ggacggaaga gttgcttgta
     2521 aaactcaatc gcgaagatct actgcgaaag cagcggactt tcgacaacgg tagcattcca
     2581 catcaaatcc acttaggcga attgcatgct atacttagaa ggcaggagga tttttatccg
     2641 ttcctcaaag acaatcgtga aaagattgag aaaatcctaa cctttcgcat accttactat
     2701 gtgggacccc tggcccgagg gaactctcgg ttcgcatgga tgacaagaaa gtccgaagaa
     2761 acgattactc catggaattt tgaggaagtt gtcgataaag gtgcgtcagc tcaatcgttc
     2821 atcgagagga tgaccaactt tgacaagaat ttaccgaacg aaaaagtatt gcctaagcac
     2881 agtttacttt acgagtattt cacagtgtac aatgaactca cgaaagttaa gtatgtcact
     2941 gagggcatgc gtaaacccgc ctttctaagc ggagaacaga agaaagcaat agtagatctg
     3001 ttattcaaga ccaaccgcaa agtgacagtt aagcaattga aagaggacta ctttaagaaa
     3061 attgaatgct tcgattctgt cgagatctcc ggggtagaag atcgatttaa tgcgtcactt
     3121 ggtacgtatc atgacctcct aaagataatt aaagataagg acttcctgga taacgaagag
     3181 aatgaagata tcttagaaga tatagtgttg actcttaccc tctttgaaga tcgggaaatg
     3241 attgaggaaa gactaaaaac atacgctcac ctgttcgacg ataaggttat gaaacagtta
     3301 aagaggcgtc gctatacggg ctggggacga ttgtcgcgga aacttatcaa cgggataaga
     3361 gacaagcaaa gtggtaaaac tattctcgat tttctaaaga gcgacggctt cgccaatagg
     3421 aactttatgc agctgatcca tgatgactct ttaaccttca aagaggatat acaaaaggca
     3481 caggtttccg gacaagggga ctcattgcac gaacatattg cgaatcttgc tggttcgcca
     3541 gccatcaaaa agggcatact ccagacagtc aaagtagtgg atgagctagt taaggtcatg
     3601 ggacgtcaca aaccggaaaa cattgtaatc gagatggcac gcgaaaatca aacgactcag
     3661 aaggggcaaa aaaacagtcg agagcggatg aagagaatag aagagggtat taaagaactg
     3721 ggcagccaga tcttaaagga gcatcctgtg gaaaataccc aattgcagaa cgagaaactt
     3781 tacctctatt acctacaaaa tggaagggac atgtatgttg atcaggaact ggacataaac
     3841 cgtttatctg attacgacgt cgatcacatt gtaccccaat cctttttgaa ggacgattca
     3901 atcgacaata aagtgcttac acgctcggat aagaaccgag ggaaaagtga caatgttcca
     3961 agcgaggaag tcgtaaagaa aatgaagaac tattggcggc agctcctaaa tgcgaaactg
     4021 ataacgcaaa gaaagttcga taacttaact aaagctgaga ggggtggctt gtctgaactt
     4081 gacaaggccg gatttattaa acgtcagctc gtggaaaccc gccaaatcac aaagcatgtt
     4141 gcacagatac tagattcccg aatgaatacg aaatacgacg agaacgataa gctgattcgg
     4201 gaagtcaaag taatcacttt aaagtcaaaa ttggtgtcgg acttcagaaa ggattttcaa
     4261 ttctataaag ttagggagat aaataactac caccatgcgc acgacgctta tcttaatgcc
     4321 gtcgtaggga ccgcactcat taagaaatac ccgaagctag aaagtgagtt tgtgtatggt
     4381 gattacaaag tttatgacgt ccgtaagatg atcgcgaaaa gcgaacagga gataggcaag
     4441 gctacagcca aatacttctt ttattctaac attatgaatt tctttaagac ggaaatcact
     4501 ctggcaaacg gagagatacg caaacgacct ttaattgaaa ccaatgggga gacaggtgaa
     4561 atcgtatggg ataagggccg ggacttcgcg acggtgagaa aagttttgtc catgccccaa
     4621 gtcaacatag taaagaaaac tgaggtgcag accggagggt tttcaaagga atcgattctt
     4681 ccaaaaagga atagtgataa gctcatcgct cgtaaaaagg actgggaccc gaaaaagtac
     4741 ggtggcttcg atagccctac agttgcctat tctgtcctag tagtggcaaa agttgagaag
     4801 ggaaaatcca agaaactgaa gtcagtcaaa gaattattgg ggataacgat tatggagcgc
     4861 tcgtcttttg aaaagaaccc catcgacttc cttgaggcga aaggttacaa ggaagtaaaa
     4921 aaggatctca taattaaact accaaagtat agtctgtttg agttagaaaa tggccgaaaa
     4981 cggatgttgg ctagcgccgg agagcttcaa aaggggaacg aactcgcact accgtctaaa
     5041 tacgtgaatt tcctgtattt agcgtcccat tacgagaagt tgaaaggttc acctgaagat
     5101 aacgaacaga agcaactttt tgttgagcag cacaaacatt atctcgacga aatcatagag
     5161 caaatttcgg aattcagtaa gagagtcatc ctagctgatg ccaatctgga caaagtatta
     5221 agcgcataca acaagcacag ggataaaccc atacgtgagc aggcggaaaa tattatccat
     5281 ttgtttactc ttaccaacct cggcgctcca gccgcattca agtattttga cacaacgata
     5341 gatcgcaaac gatacacttc taccaaggag gtgctagacg cgacactgat tcaccaatcc
     5401 atcacgggat tatatgaaac tcggatagat ttgtcacagc ttgggggtga ctctggtggt
     5461 tctactaatc tgtcagatat tattgaaaag gagaccggta agcaactggt tatccaggaa
     5521 tccatcctca tgctcccaga ggaggtggaa gaagtcattg ggaacaagcc ggaaagcgat
     5581 atactcgtgc acaccgccta cgacgagagc accgacgaga atgtcatgct tctgactagc
     5641 gacgcccctg aatacaagcc ttgggctctg gtcatacagg atagcaacgg tgagaacaag
     5701 attaagatgc tctctggtgg ttctcccaag aagaagagga aagtctaacc ggtcatcatc
     5761 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     5821 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5881 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5941 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     6001 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac
     6061 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6121 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg
     6181 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6241 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6301 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6361 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6421 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6481 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6541 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6601 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6661 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6721 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6781 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6841 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6901 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6961 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7021 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7081 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7141 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7201 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7261 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7321 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7381 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7441 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7501 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7561 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7621 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7681 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7741 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7801 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7861 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7921 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7981 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8041 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8101 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8161 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8221 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc
     8281 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     8341 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     8401 ggcaaggctt gaccg
//