LOCUS       Exported                8532 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses mA1-BE3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-mA1-BE3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8532)
  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 8532)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8532
                     /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             1144..5244
                     /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             5257..5505
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5518..5538
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5547..5564
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5587..5604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5593..5817
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5888..5904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5888..5904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5901..5923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5912..5928
                     /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(5936..5966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5981..6002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6119..6136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6290..6878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6370..6389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7049..7909)
                     /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     7672..7691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7910..8014)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8093..8112)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8284..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 gagttccgag
      421 acaggccctg tagctgttga tcccactctg aggagaagaa ttgagcccca cgagtttgaa
      481 gtcttctttg acccccggga gcttcggaaa gagacctgtc tgctgtatga gatcaactgg
      541 ggtggaaggc acagtgtctg gcgacacacg agccaaaaca ccagcaacca cgttgaagtc
      601 aacttcttag aaaaatttac tacagaaaga tactttcgtc cgaacaccag atgctccatt
      661 acctggttcc tgtcctggag tccctgcggg gagtgctcca gggccattac agagtttctg
      721 agccgacacc cctatgtaac tctgtttatt tacatagcac ggctttatca ccacacggat
      781 cagcgaaacc gccaaggact cagggacctt attagcagcg gtgtgactat ccagatcatg
      841 acagagcaag agtattgtta ctgctggagg aatttcgtca actacccccc ttcaaacgaa
      901 gcttattggc caaggtaccc ccatctgtgg gtgaaactgt atgtactgga gctctactgc
      961 atcattttag gacttccacc ctgtttaaaa attttaagaa gaaagcaacc tcaactcacg
     1021 tttttcacaa ttactcttca aacctgccat taccaaagga taccacccca tctcctttgg
     1081 gctacagggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa
     1141 agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc
     1201 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt
     1261 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag
     1321 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt
     1381 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt
     1441 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga
     1501 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa
     1561 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat
     1621 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat
     1681 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct
     1741 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga
     1801 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac
     1861 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa
     1921 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca
     1981 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc
     2041 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca
     2101 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt
     2161 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca
     2221 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta
     2281 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga
     2341 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat
     2401 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt
     2461 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct
     2521 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa
     2581 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag
     2641 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg
     2701 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta
     2761 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca
     2821 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc
     2881 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata
     2941 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg
     3001 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct
     3061 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga
     3121 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc
     3181 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac
     3241 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg
     3301 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca
     3361 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta
     3421 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg
     3481 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct
     3541 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg
     3601 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac
     3661 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg
     3721 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag
     3781 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta
     3841 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag
     3901 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat
     3961 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca
     4021 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac
     4081 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa
     4141 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag
     4201 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct
     4261 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga
     4321 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc
     4381 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg
     4441 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc
     4501 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc
     4561 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc
     4621 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac
     4681 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag
     4741 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt
     4801 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc
     4861 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag
     4921 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc
     4981 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa
     5041 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct
     5101 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag
     5161 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata
     5221 gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa
     5281 aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg
     5341 gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag
     5401 agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct
     5461 ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc
     5521 aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct
     5581 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5641 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5701 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5761 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     5821 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt
     5881 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5941 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat
     6001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6121 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6181 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6241 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6301 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6361 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6421 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6481 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6541 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6601 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6661 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6721 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6781 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6841 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6901 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6961 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7021 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7081 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7141 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7201 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7261 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7321 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7381 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7441 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7501 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7561 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7621 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7681 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7741 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7801 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7861 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7921 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7981 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     8041 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct
     8101 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     8161 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     8221 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     8281 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     8341 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     8401 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     8461 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     8521 atcaagtgta tc
//
