LOCUS       sequence-368592-        8109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DddA E1347A variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8109)
  AUTHORS   Mok YG, Lee JM, Chung E, Lee J, Lim K, Cho SI, Kim JS
  TITLE     Base editing in human cells with monomeric DddA-TALE fusion 
            deaminases.
  JOURNAL   Nat Commun. 2022 Jul 12;13(1):4038. doi: 10.1038/s41467-022-31745-y.
  PUBMED    35821233
REFERENCE   2  (bases 1 to 8109)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-07-12"; volume: "13(1):4038. doi"; pages: " 
            10.1038/s41467-022-31745-y"
COMMENT     Sequence Label: pCMV-E1347A-nCas9(D10A)-UGI
FEATURES             Location/Qualifiers
     source          1..8109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1372..1395,1396..1398,1399..5472)
                     /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             5485..5733
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5746..5766
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5770..5796
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             5770..5796
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(5827..5845)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5828..5845)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5865..5882)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5871..6095
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6183..6200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6354..6942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6434..6453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7113..7904,7905..7973))
                     /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     7736..7755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7974..8078)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc
      901 atgggatccg gcagctacgc cctgggtccg tatcagatta gcgccccgca gctgccagct
      961 tacaatggtc agaccgtggg taccttctac tatgtgaacg acgcgggcgg tctggagagc
     1021 aaggtgttta gcagcggcgg tccaaccccg tacccaaact atgccaatgc cggtcatgtg
     1081 gccggtcaga gcgccctgtt catgcgtgat aacggcatca gcgagggtct ggtgttccac
     1141 aacaacccgg aaggcacctg cggtttttgc gtgaacatga ccgagaccct gctgccggaa
     1201 aacgcgaaaa tgaccgtggt gccgccggaa ggtgccattc cagtgaagcg cggcgctacc
     1261 ggtgaaacca aagtgtttac cggtaacagc aacagcccga agagcccgac caaaggcggt
     1321 tgcagcggca gcgagactcc cgggacctca gagtccgcca cacccgaaag tgacaagaag
     1381 tacagcatcg gcctggccat cggtaccaac agcgtgggct gggccgtgat caccgacgag
     1441 tacaaggtgc ccagcaagaa gttcaaggtg ctgggcaaca ccgaccgcca cagcatcaag
     1501 aagaacctga tcggcgccct gctgttcgac agcggcgaga ccgccgaggc cacccgcctg
     1561 aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctgcaggag
     1621 atcttcagca acgagatggc caaggtggac gacagcttct tccaccgcct ggaggagagc
     1681 ttcctggtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtggac
     1741 gaggtggcct accacgagaa gtaccccacc atctaccacc tgcgcaagaa gctggtggac
     1801 agcaccgaca aggccgacct gcgcctgatc tacctggccc tggcccacat gatcaagttc
     1861 cgcggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1921 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg agaaccccat caacgccagc
     1981 ggcgtggacg ccaaggccat cctgagcgcc cgcctgagca agagccgccg cctggagaac
     2041 ctgatcgccc agctgcccgg cgagaagaag aacggcctgt tcggcaacct gatcgccctg
     2101 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga cgccaagctg
     2161 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2221 cagtacgccg acctgttcct ggccgccaag aacctgagcg acgccatcct gctgagcgac
     2281 atcctgcgcg tgaacaccga gatcaccaag gcccccctga gcgccagcat gatcaagcgc
     2341 tacgacgagc accaccagga cctgaccctg ctgaaggccc tggtgcgcca gcagctgccc
     2401 gagaagtaca aggagatctt cttcgaccag agcaagaacg gctacgccgg ctacatcgac
     2461 ggcggcgcca gccaggagga gttctacaag ttcatcaagc ccatcctgga gaagatggac
     2521 ggcaccgagg agctgctggt gaagctgaac cgcgaggacc tgctgcgcaa gcagcgcacc
     2581 ttcgacaacg gcagcatccc ccaccagatc cacctgggcg agctgcacgc catcctgcgc
     2641 cgccaggagg acttctaccc cttcctgaag gacaaccgcg agaagatcga gaagatcctg
     2701 accttccgca tcccctacta cgtgggcccc ctggcccgcg gcaacagccg cttcgcctgg
     2761 atgacccgca agagcgagga gaccatcacc ccctggaact tcgaggaggt ggtggacaag
     2821 ggcgccagcg cccagagctt catcgagcgc atgaccaact tcgacaagaa cctgcccaac
     2881 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta caacgagctg
     2941 accaaggtga agtacgtgac cgagggcatg cgcaagcccg ccttcctgag cggcgagcag
     3001 aagaaggcca tcgtggacct gctgttcaag accaaccgca aggtgaccgt gaagcagctg
     3061 aaggaggact acttcaagaa gatcgagtgc ttcgacagcg tggagatcag cggcgtggag
     3121 gaccgcttca acgccagcct gggcacctac cacgacctgc tgaagatcat caaggacaag
     3181 gacttcctgg acaacgagga gaacgaggac atcctggagg acatcgtgct gaccctgacc
     3241 ctgttcgagg accgcgagat gatcgaggag cgcctgaaga cctacgccca cctgttcgac
     3301 gacaaggtga tgaagcagct gaagcgccgc cgctacaccg gctggggccg cctgagccgc
     3361 aagcttatca acggcatccg cgacaagcag agcggcaaga ccatcctgga cttcctgaag
     3421 agcgacggct tcgccaaccg caacttcatg cagctgatcc acgacgacag cctgaccttc
     3481 aaggaggaca tccagaaggc ccaggtgagc ggccagggcg acagcctgca cgagcacatc
     3541 gccaacctgg ccggcagccc cgccatcaag aagggcatcc tgcagaccgt gaaggtggtg
     3601 gacgagctgg tgaaggtgat gggccgccac aagcccgaga acatcgtgat cgagatggcc
     3661 cgcgagaacc agaccaccca gaagggccag aagaacagcc gcgagcgcat gaagcgcatc
     3721 gaggagggca tcaaggagct gggcagccag atcctgaagg agcaccccgt ggagaacacc
     3781 cagctgcaga acgagaagct gtacctgtac tacctgcaga acggccgcga catgtacgtg
     3841 gaccaggagc tggacatcaa ccgcctgagc gactacgacg tggaccacat cgtgccccag
     3901 agcttcctga aggacgacag catcgacaac aaggtgctga cccgcagcga caagaaccgc
     3961 ggcaagagcg acaacgtgcc cagcgaggag gtggtgaaga agatgaagaa ctactggcgc
     4021 cagctgctga acgccaagct gatcacccag cgcaagttcg acaacctgac caaggccgag
     4081 cgcggcggcc tgagcgagct ggacaaggcc ggcttcatca agcgccagct ggtggagacc
     4141 cgccagatca ccaagcacgt ggcccagatc ctggacagcc gcatgaacac caagtacgac
     4201 gagaacgaca agctgatccg cgaggtgaag gtgatcaccc tgaagagcaa gctggtgagc
     4261 gacttccgca aggacttcca gttctacaag gtgcgcgaga tcaacaacta ccaccacgcc
     4321 cacgacgcct acctgaacgc cgtggtgggc accgccctga tcaagaagta ccccaagctg
     4381 gagagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcgcaagat gatcgccaag
     4441 agcgagcagg agatcggcaa ggccaccgcc aagtacttct tctacagcaa catcatgaac
     4501 ttcttcaaga ccgagatcac cctggccaac ggcgagatcc gcaagcgccc cctgatcgag
     4561 accaacggcg agaccggcga gatcgtgtgg gacaagggcc gcgacttcgc caccgtgcgc
     4621 aaggtgctga gcatgcccca ggtgaacatc gtgaagaaga ccgaggtgca gaccggcggc
     4681 ttcagcaagg agagcatcct gcccaagcgc aacagcgaca agctgatcgc ccgcaagaag
     4741 gactgggacc ccaagaagta cggcggcttc gacagcccca ccgtggccta cagcgtgctg
     4801 gtggtggcca aggtggagaa gggcaagagc aagaagctga agagcgtgaa ggagctgctg
     4861 ggcatcacca tcatggagcg cagcagcttc gagaagaacc ccatcgactt cctggaggcc
     4921 aagggctaca aggaggtgaa gaaggacctg atcatcaagc tgcccaagta cagcctgttc
     4981 gagctggaga acggccgcaa gcgcatgctg gccagcgccg gcgagctgca gaagggcaac
     5041 gagctggccc tgcccagcaa gtacgtgaac ttcctgtacc tggccagcca ctacgagaag
     5101 ctgaagggca gccccgagga caacgagcag aagcagctgt tcgtggagca gcacaagcac
     5161 tacctggacg agatcatcga gcagatcagc gagttcagca agcgcgtgat cctggccgac
     5221 gccaacctgg acaaggtgct gagcgcctac aacaagcacc gcgacaagcc catccgcgag
     5281 caggccgaga acatcatcca cctgttcacc ctgaccaacc tgggcgcccc cgccgccttc
     5341 aagtacttcg acaccaccat cgaccgcaag cgctacacca gcaccaagga ggtgctggac
     5401 gccaccctga tccaccagag catcaccggt ctgtacgaga cccgcatcga cctgagccag
     5461 ctgggcggcg actctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt
     5521 aagcaactgg ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt
     5581 gggaacaagc cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag
     5641 aatgtcatgc ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag
     5701 gatagcaacg gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg
     5761 aaagtcgtat acccctacga cgtgcccgac tacgcctaac tcgagcatgc atctagaggg
     5821 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     5881 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     5941 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     6001 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     6061 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     6121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6181 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6241 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6301 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6361 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6421 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6481 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6541 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6601 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6661 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6721 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6781 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6841 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6901 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6961 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7021 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7081 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7141 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7201 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7261 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7321 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7381 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7441 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7501 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7561 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7621 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7681 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7741 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7801 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7861 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7921 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7981 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8041 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8101 cctgacgtc
//
