LOCUS       sequence-368591-        8109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DddA GSVG 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-UGI-nCas9(H840A)-GSVG
FEATURES             Location/Qualifiers
     source          1..8109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        82..461
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        462..665
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     616..636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     710..729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        710..728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             757..783
                     /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             757..783
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             784..804
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             820..1068
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             join(1081..3594,3595..3597,3598..5181)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     promoter        complement(5674..5692)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5675..5692)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5712..5729)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5718..5942
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6030..6047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6201..6789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6281..6300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6960..7751,7752..7820))
                     /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     7583..7602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7821..7925)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8000..8019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
       61 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      121 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      181 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      241 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      301 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      361 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      421 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      481 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      541 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      601 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      661 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      721 actataggga gacccaagct tgccaccatg ggatcctacc cctacgacgt gcccgactac
      781 gcccccaaga agaagaggaa agtcgtatct ggtggttcta ctaatctgtc agatattatt
      841 gaaaaggaga ccggtaagca actggttatc caggaatcca tcctcatgct cccagaggag
      901 gtggaagaag tcattgggaa caagccggaa agcgatatac tcgtgcacac cgcctacgac
      961 gagagcaccg acgagaatgt catgcttctg actagcgacg cccctgaata caagccttgg
     1021 gctctggtca tacaggatag caacggtgag aacaagatta agatgctctc tggtggttct
     1081 gacaagaagt acagcatcgg cctggacatc ggtaccaaca gcgtgggctg ggccgtgatc
     1141 accgacgagt acaaggtgcc cagcaagaag ttcaaggtgc tgggcaacac cgaccgccac
     1201 agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggcgagac cgccgaggcc
     1261 acccgcctga agcgcaccgc ccgccgccgc tacacccgcc gcaagaaccg catctgctac
     1321 ctgcaggaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccaccgcctg
     1381 gaggagagct tcctggtgga ggaggacaag aagcacgagc gccaccccat cttcggcaac
     1441 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gcgcaagaag
     1501 ctggtggaca gcaccgacaa ggccgacctg cgcctgatct acctggccct ggcccacatg
     1561 atcaagttcc gcggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1621 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga gaaccccatc
     1681 aacgccagcg gcgtggacgc caaggccatc ctgagcgccc gcctgagcaa gagccgccgc
     1741 ctggagaacc tgatcgccca gctgcccggc gagaagaaga acggcctgtt cggcaacctg
     1801 atcgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggac
     1861 gccaagctgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     1921 atcggcgacc agtacgccga cctgttcctg gccgccaaga acctgagcga cgccatcctg
     1981 ctgagcgaca tcctgcgcgt gaacaccgag atcaccaagg cccccctgag cgccagcatg
     2041 atcaagcgct acgacgagca ccaccaggac ctgaccctgc tgaaggccct ggtgcgccag
     2101 cagctgcccg agaagtacaa ggagatcttc ttcgaccaga gcaagaacgg ctacgccggc
     2161 tacatcgacg gcggcgccag ccaggaggag ttctacaagt tcatcaagcc catcctggag
     2221 aagatggacg gcaccgagga gctgctggtg aagctgaacc gcgaggacct gctgcgcaag
     2281 cagcgcacct tcgacaacgg cagcatcccc caccagatcc acctgggcga gctgcacgcc
     2341 atcctgcgcc gccaggagga cttctacccc ttcctgaagg acaaccgcga gaagatcgag
     2401 aagatcctga ccttccgcat cccctactac gtgggccccc tggcccgcgg caacagccgc
     2461 ttcgcctgga tgacccgcaa gagcgaggag accatcaccc cctggaactt cgaggaggtg
     2521 gtggacaagg gcgccagcgc ccagagcttc atcgagcgca tgaccaactt cgacaagaac
     2581 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtac
     2641 aacgagctga ccaaggtgaa gtacgtgacc gagggcatgc gcaagcccgc cttcctgagc
     2701 ggcgagcaga agaaggccat cgtggacctg ctgttcaaga ccaaccgcaa ggtgaccgtg
     2761 aagcagctga aggaggacta cttcaagaag atcgagtgct tcgacagcgt ggagatcagc
     2821 ggcgtggagg accgcttcaa cgccagcctg ggcacctacc acgacctgct gaagatcatc
     2881 aaggacaagg acttcctgga caacgaggag aacgaggaca tcctggagga catcgtgctg
     2941 accctgaccc tgttcgagga ccgcgagatg atcgaggagc gcctgaagac ctacgcccac
     3001 ctgttcgacg acaaggtgat gaagcagctg aagcgccgcc gctacaccgg ctggggccgc
     3061 ctgagccgca agcttatcaa cggcatccgc gacaagcaga gcggcaagac catcctggac
     3121 ttcctgaaga gcgacggctt cgccaaccgc aacttcatgc agctgatcca cgacgacagc
     3181 ctgaccttca aggaggacat ccagaaggcc caggtgagcg gccagggcga cagcctgcac
     3241 gagcacatcg ccaacctggc cggcagcccc gccatcaaga agggcatcct gcagaccgtg
     3301 aaggtggtgg acgagctggt gaaggtgatg ggccgccaca agcccgagaa catcgtgatc
     3361 gagatggccc gcgagaacca gaccacccag aagggccaga agaacagccg cgagcgcatg
     3421 aagcgcatcg aggagggcat caaggagctg ggcagccaga tcctgaagga gcaccccgtg
     3481 gagaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa cggccgcgac
     3541 atgtacgtgg accaggagct ggacatcaac cgcctgagcg actacgacgt ggacgccatc
     3601 gtgccccaga gcttcctgaa ggacgacagc atcgacaaca aggtgctgac ccgcagcgac
     3661 aagaaccgcg gcaagagcga caacgtgccc agcgaggagg tggtgaagaa gatgaagaac
     3721 tactggcgcc agctgctgaa cgccaagctg atcacccagc gcaagttcga caacctgacc
     3781 aaggccgagc gcggcggcct gagcgagctg gacaaggccg gcttcatcaa gcgccagctg
     3841 gtggagaccc gccagatcac caagcacgtg gcccagatcc tggacagccg catgaacacc
     3901 aagtacgacg agaacgacaa gctgatccgc gaggtgaagg tgatcaccct gaagagcaag
     3961 ctggtgagcg acttccgcaa ggacttccag ttctacaagg tgcgcgagat caacaactac
     4021 caccacgccc acgacgccta cctgaacgcc gtggtgggca ccgccctgat caagaagtac
     4081 cccaagctgg agagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcgcaagatg
     4141 atcgccaaga gcgagcagga gatcggcaag gccaccgcca agtacttctt ctacagcaac
     4201 atcatgaact tcttcaagac cgagatcacc ctggccaacg gcgagatccg caagcgcccc
     4261 ctgatcgaga ccaacggcga gaccggcgag atcgtgtggg acaagggccg cgacttcgcc
     4321 accgtgcgca aggtgctgag catgccccag gtgaacatcg tgaagaagac cgaggtgcag
     4381 accggcggct tcagcaagga gagcatcctg cccaagcgca acagcgacaa gctgatcgcc
     4441 cgcaagaagg actgggaccc caagaagtac ggcggcttcg acagccccac cgtggcctac
     4501 agcgtgctgg tggtggccaa ggtggagaag ggcaagagca agaagctgaa gagcgtgaag
     4561 gagctgctgg gcatcaccat catggagcgc agcagcttcg agaagaaccc catcgacttc
     4621 ctggaggcca agggctacaa ggaggtgaag aaggacctga tcatcaagct gcccaagtac
     4681 agcctgttcg agctggagaa cggccgcaag cgcatgctgg ccagcgccgg cgagctgcag
     4741 aagggcaacg agctggccct gcccagcaag tacgtgaact tcctgtacct ggccagccac
     4801 tacgagaagc tgaagggcag ccccgaggac aacgagcaga agcagctgtt cgtggagcag
     4861 cacaagcact acctggacga gatcatcgag cagatcagcg agttcagcaa gcgcgtgatc
     4921 ctggccgacg ccaacctgga caaggtgctg agcgcctaca acaagcaccg cgacaagccc
     4981 atccgcgagc aggccgagaa catcatccac ctgttcaccc tgaccaacct gggcgccccc
     5041 gccgccttca agtacttcga caccaccatc gaccgcaagc gctacaccag caccaaggag
     5101 gtgctggacg ccaccctgat ccaccagagc atcaccggtc tgtacgagac ccgcatcgac
     5161 ctgagccagc tgggcggcga cagcggcagc gagactcccg ggacctcaga gtccgccaca
     5221 cccgaaagtg gcagctacgc cctgggtccg tatcagatta gcgccccgca gctgccagca
     5281 tacaatggtc agaccgtggg taccttctac tatgtgaacg acgctggcgg tctggagggc
     5341 aaggtgttta gcagcggcgg tccaaccccg tacccaaact atgccaatgc cggtcatgtg
     5401 gagagtcaga gcgccctgtt catgcgtgat aacggcatca gcgagggtct ggtgttccac
     5461 aacaacccgg aaggcacctg cggtttttgc gtgaacatga ccgagaccct gctgccggaa
     5521 aacgcgaaaa tgaccgtggt gccgccggaa ggtgtcattc cagtgaagcg cggcgctacc
     5581 ggtgaaacca aagtgtttac cggtaacagc aacggcccga agagcccgac caaaggcggt
     5641 tgctaactcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc
     5701 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5761 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5821 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5881 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     5941 ggcttctgag gcggaaagaa ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6001 ggtttgcgta ttgggcgctc tttcgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6061 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7561 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7921 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     7981 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     8041 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     8101 ggcaaggct
//
