LOCUS       sequence-422063-        8580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of base editor enCas12a-ABE8e-V106W in mammalian cells 
            for base editing, along with non-fused EGFP fluorescence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8580)
  AUTHORS   Chen F, Lian M, Ma B, Gou S, Luo X, Yang K, Shi H, Xie J, Ge W, 
            Ouyang Z, Lai C, Li N, Zhang Q, Jin Q, Liang Y, Chen T, Wang J, Zhao
            X, Li L, Yu M, Ye Y, Wang K, Wu H, Lai L
  TITLE     Multiplexed base editing through Cas12a variant-mediated cytosine 
            and adenine base editors.
  JOURNAL   Commun Biol. 2022 Nov 2;5(1):1163. doi: 10.1038/s42003-022-04152-8.
  PUBMED    36323848
REFERENCE   2  (bases 1 to 8580)
  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: "Commun 
            Biol."; date: "2022-11-2"; volume: "5(1):1163. doi"; pages: " 
            10.1038/s42003-022-04152-8"
COMMENT     Sequence Label: pCMV-enCas12a-ABE8e-V106W
FEATURES             Location/Qualifiers
     source          1..8580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4789..4809
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4819..4878
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             join(4879..4881,4882..5595)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4879..4881,4882..5592)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4921..4942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5182..5201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5529..5550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5635..5652)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5641..5865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5936..5952)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5936..5952)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5949..5971)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5960..5976
                     /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(join(5984..5990,5991..6008,6009..6014))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6029..6050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6167..6184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6338..6926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6418..6437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7097..7888,7889..7957))
                     /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     7720..7739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7958..8062)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8141..8160)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8332..8580,1..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 gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg cgatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggctgg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcat caaagctgga gaaattcacc
     1081 aactgttata gcctgtctaa gaccctgcgc ttcaaggcaa tcccagtggg caagacacaa
     1141 gagaacattg acaacaaacg gctcctggtg gaggatgaga agagggctga agattacaag
     1201 ggcgttaaga agctgctgga taggtactat ctgtcattca tcaacgatgt cctccacagt
     1261 atcaagctga agaatctgaa caattacatt tctctgttcc ggaagaagac acggaccgag
     1321 aaggagaaca aagagctgga gaatctggag atcaacctgc ggaaggagat cgccaaggcc
     1381 ttcaagggca acgagggcta caagtccctg tttaagaagg atatcatcga gacaatcctg
     1441 ccagagttcc tggacgataa ggacgagatc gccctggtga acagcttcaa tggctttacc
     1501 acagccttca ccggcttctt taggaacaga gagaatatgt tttccgagga ggccaagagc
     1561 acatccatcg ccttcaggtg tatcaacgag aatctgaccc gctacatctc taatatggac
     1621 atcttcgaga aggtggacgc catctttgat aagcacgagg tgcaggagat caaggagaag
     1681 atcctgaaca gcgactatga tgtggaggat ttctttgagg gcgagttctt taactttgtg
     1741 ctgacacagg agggcatcga cgtgtataac gccatcatcg gcggcttcgt gaccgagagc
     1801 ggcgagaaga tcaagggcct gaacgagtac atcaacctgt ataatcagaa aaccaagcag
     1861 aagctgccta agtttaagcc actgtataag caggtgctga gcgatcggga gtctctgagc
     1921 ttctacggcg agggctatac atccgatgag gaggtgctgg aggtgtttag aaacaccctg
     1981 aacaagaaca gcgagatctt cagctccatc aagaagctgg agaagctgtt caagaatttt
     2041 gacgagtact ctagcgccgg catctttgtg aagaacggcc ccgccatcag cacaatctcc
     2101 aaggatatct tcggcgagtg gaacgtgatc cgggacaagt ggaatgccga gtatgacgat
     2161 atccacctga agaagaaggc cgtggtgacc gagaagtacg aggacgatcg gagaaagtcc
     2221 ttcaagaaga tcggctcctt ttctctggag cagctgcagg agtacgccga cgccgatctg
     2281 tctgtggtgg agaagctgaa ggagatcatc atccagaagg tggatgagat ctacaaggtg
     2341 tatggctcct ctgagaagct gttcgacgcc gattttgtgc tggagaagag cctgaagaag
     2401 aacgacgccg tggtggccat catgaaggac ctgctggatt ctgtgaagag cttcgagaat
     2461 tacatcaagg ccttctttgg cgagggcaag gagacaaaca gggacgagtc cttctatggc
     2521 gattttgtgc tggcctacga catcctgctg aaggtggacc acatctacga tgccatccgc
     2581 aattatgtga cccagaagcc ctactctaag gataagttca agctgtattt tcagaaccct
     2641 cagttcatga ggggctggga caaggatagg gagacagact atcgggccac catcctgaga
     2701 tacggctcca agtactatct ggccatcatg gataagaagt acgccaagtg cctgcagaag
     2761 atcgacaagg acgatgtgaa cggcaattac gagaagatca actataagct gctgcccggc
     2821 cctaataaga tgctgccaaa ggtgttcttt tctaagaagt ggatggccta ctataacccc
     2881 agcgaggaca tccagaagat ctacaagaat ggcacattca agaagggcga tatgtttaac
     2941 ctgaatgact gtcacaagct gatcgacttc tttaaggata gcatctcccg gtatccaaag
     3001 tggtccaatg catacgattt caacttcagc gagactgaga agtacaaaga catcgctggc
     3061 ttctaccggg aggtggaaga gcaaggctat aaggtgtcat tcgaatccgc ttctaagaag
     3121 gaagtggata agctcgtgga agagggtaag ctgtacatgt tccagatata caacaaagac
     3181 ttcagcgata agagccacgg cactccaaac ctccatacta tgtatttcaa gctgctgttt
     3241 gacgagaaca accacggaca gattaggctg tcaggaggcg cagaactctt catgcgcaga
     3301 gcttcactga agaaggagga actcgttgtc cacccagcca atagccctat agccaataag
     3361 aatccagaca atcctaagaa aaccactact ctgtcttacg atgtgtataa ggataagaga
     3421 ttctctgaag atcagtacga actgcacata cccattgcca ttaacaagtg ccctaagaac
     3481 atcttcaaga ttaacacaga ggttagagtg ctcctgaaac acgacgataa cccttatgtt
     3541 ataggcattg ctcgcggaga gagaaacctg ctgtacatcg tcgtggtgga cggcaaaggc
     3601 aacatcgtgg aacagtacag tctcaatgaa atcattaaca atttcaacgg aatccgcatt
     3661 aagaccgact accattctct cctcgacaag aaggagaaag aaaggttcga agcaagacag
     3721 aattggacaa gtatagagaa tatcaaagaa ctgaaggctg ggtacatctc tcaggttgtg
     3781 cacaagatat gtgagctggt ggagaagtac gacgctgtta tcgccctcgc ggacctgaat
     3841 agcggcttca agaactccag ggtgaaggtg gagaagcagg tgtatcagaa gttcgagaag
     3901 atgctgatcg acaagctcaa ctatatggtg gacaagaaat ccaatccttg cgctactggt
     3961 ggagccctga agggctatca aatcaccaat aagttcgaat ctttcaagtc tatgagcacc
     4021 cagaatggct tcatcttcta catacccgca tggctgacat ccaagattga tccctctacc
     4081 ggatttgtta atctgctcaa gactaagtac acctctattg ctgactcaaa gaagttcata
     4141 tcatcatttg accgcatcat gtacgtgcca gaagaggacc tgttcgagtt tgccctggat
     4201 tacaagaatt tctctcggac tgacgccgac tacatcaaga agtggaagct ctactcttat
     4261 ggtaatcgga ttcgcatatt ccgcaatccc aagaagaata acgtgttcga ttgggaggaa
     4321 gtttgcctca ccagcgctta caaggagctg ttcaataagt atgggattaa ctaccagcag
     4381 ggcgacataa gagccctgct gtgcgaacaa tctgataagg cattctattc ctctttcatg
     4441 gcactgatgt cactgatgct gcaaatgcgc aattccatca ccggaagaac agacgtggcc
     4501 tttctgatct ctcctgtcaa gaactcagat ggcatcttct acgattcccg caactatgaa
     4561 gcacaggaga atgctatcct gcctaagaat gccgatgcaa atggagccta taacatcgcc
     4621 agaaaggtcc tctgggccat aggacaattc aagaaagctg aagatgagaa gctggacaag
     4681 gtgaagatcg ccatttcaaa caaagagtgg ctcgaatatg ctcagacctc agtgaagcat
     4741 ggatcatctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     4801 aggaaagtcg gctcgggcca gtgtactaat tatgctctct tgaaattggc tggagatgtt
     4861 gagagcaacc caggtcccgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4921 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4981 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     5041 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     5101 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     5161 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     5221 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     5281 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     5341 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     5401 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     5461 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     5521 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     5581 gagctgtaca agtaaatcat caccatcacc attgagttta aacccgctga tcagcctcga
     5641 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5701 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5761 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5821 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5881 gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5941 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6001 gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6061 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6121 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6181 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6241 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6301 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6361 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6421 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6481 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6541 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6601 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6661 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6721 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6781 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6841 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6901 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca
     6961 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7021 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7081 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7141 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7201 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7261 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7321 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7381 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7441 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7501 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7561 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7621 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7681 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7741 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7801 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7861 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7921 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7981 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     8101 cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg
     8161 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     8221 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     8281 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     8341 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     8401 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     8461 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     8521 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
//
