LOCUS       sequence-422133-        8580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of base editor RR-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-RR-ABE8e-V106W
FEATURES             Location/Qualifiers
     source          1..8580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3298..3318
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3328..3387
                     /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(3388..3390,3391..4104)
                     /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(3388..3390,3391..4101)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3430..3451)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3691..3710)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4038..4059
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4144..4161)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4150..4374
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4445..4461)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4445..4461)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4458..4480)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4469..4485
                     /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(4493..4499,4500..4517,4518..4523))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4538..4559
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4676..4693)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4847..5435)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4927..4946)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5606..6397,6398..6466))
                     /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     6229..6248
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6467..6571)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6650..6669)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6841..7220
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7221..7424
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7375..7395
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7466..7485
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7466..7484
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7534..7554
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gggtttacca cagcctttac cggcttcttc gacaatagag agaatatgtt ctctgaagag
       61 gccaaatcca ctagcatcgc ctttcgctgc ataaacgaga acctgactag gtacatcagc
      121 aatatggaca tctttgagaa agtcgatgcc atattcgaca aacatgaggt gcaggagatt
      181 aaggagaaga tcctgaactc agattacgat gtcgaagatt tcttcgaggg agagttcttc
      241 aacttcgtgc tcacacaaga gggcattgat gtgtacaatg caatcattgg agggttcgtg
      301 acagagagtg gcgagaagat aaagggcctg aacgagtata tcaacctcta caaccagaaa
      361 accaagcaga aactgcctaa gttcaagcca ctgtacaaac aagtgctctc agatagggaa
      421 agcctgagct tctacggtga agggtataca tcagatgaag aagtgctcga agtgttccgc
      481 aacaccctca ataagaacag tgaaatcttc tcttcaatca agaagctgga gaaactgttc
      541 aagaatttcg atgagtactc ctctgccgga atctttgtga agaatggccc tgcaatatcc
      601 actattagca aagacatctt tggcgagtgg aacgttatca gggataagtg gaatgccgag
      661 tacgatgata ttcatctcaa gaagaaagcc gtggttacag agaaatacga ggatgataga
      721 cgcaagagct ttaagaagat tggtagcttc tctctcgaac agctgcagga gtacgccgac
      781 gctgacctgt cagtcgtgga gaaactcaag gagatcataa tccagaaggt ggatgaaatc
      841 tacaaagtgt atggaagctc tgagaaactc ttcgatgcag actttgttct ggagaagagt
      901 ctgaagaaga acgacgcagt ggttgctatc atgaaggacc tgctggattc tgttaagtct
      961 ttcgagaatt acattaaggc attctttggt gaagggaagg agacaaatag ggacgagagc
     1021 ttctatggcg actttgttct ggcctacgac atcctcctca aggttgacca catctatgac
     1081 gctatacgga attacgttac ccagaagccc tatagcaaag acaagttcaa gctgtatttc
     1141 cagaatccac agtttatgag ggggtgggat aaagacaaag aaacagatta cagggccact
     1201 atcctgcggt acggcagcaa atactatctg gctatcatgg ataagaagta cgccaaatgc
     1261 ctccagaaga tcgacaagga cgacgtgaac ggtaactacg agaagatcaa ttacaagctc
     1321 ctgccaggac ctaacaagat gctgccccgc gtgttcttct ccaagaaatg gatggcctac
     1381 tataacccaa gcgaggacat tcagaagata tacaagaatg ggacattcaa gaagggcgat
     1441 atgttcaacc tcaacgactg ccacaagctg attgatttct tcaaggatag catttctcgc
     1501 tatcccaagt ggtctaatgc atacgatttc aacttcagcg agactgagaa gtacaaagac
     1561 atcgctggct tctaccggga ggtggaagag caaggctata aggtgtcatt cgaatccgct
     1621 tctaagaagg aagtggataa gctcgtggaa gagggtaagc tgtacatgtt ccagatatac
     1681 aacaaagact tcagcgataa gagccacggc actccaaacc tccatactat gtatttcaag
     1741 ctgctgtttg acgagaacaa ccacggacag attaggctgt caggaggcgc agaactcttc
     1801 atgcgcagag cttcactgaa gaaggaggaa ctcgttgtcc acccagccaa tagccctata
     1861 gccaataaga atccagacaa tcctaagaaa accactactc tgtcttacga tgtgtataag
     1921 gataagagat tctctgaaga tcagtacgaa ctgcacatac ccattgccat taacaagtgc
     1981 cctaagaaca tcttcaagat taacacagag gttagagtgc tcctgaaaca cgacgataac
     2041 ccttatgtta taggcattgc tcgcggagag agaaacctgc tgtacatcgt cgtggtggac
     2101 ggcaaaggca acatcgtgga acagtacagt ctcaatgaaa tcattaacaa tttcaacgga
     2161 atccgcatta agaccgacta ccattctctc ctcgacaaga aggagaaaga aaggttcgaa
     2221 gcaagacaga attggacaag tatagagaat atcaaagaac tgaaggctgg gtacatctct
     2281 caggttgtgc acaagatatg tgagctggtg gagaagtacg acgctgttat cgccctcgcg
     2341 gacctgaata gcggcttcaa gaactccagg gtgaaggtgg agaagcaggt gtatcagaag
     2401 ttcgagaaga tgctgatcga caagctcaac tatatggtgg acaagaaatc caatccttgc
     2461 gctactggtg gagccctgaa gggctatcaa atcaccaata agttcgaatc tttcaagtct
     2521 atgagcaccc agaatggctt catcttctac atacccgcat ggctgacatc caagattgat
     2581 ccctctaccg gatttgttaa tctgctcaag actaagtaca cctctattgc tgactcaaag
     2641 aagttcatat catcatttga ccgcatcatg tacgtgccag aagaggacct gttcgagttt
     2701 gccctggatt acaagaattt ctctcggact gacgccgact acatcaagaa gtggaagctc
     2761 tactcttatg gtaatcggat tcgcatattc cgcaatccca agaagaataa cgtgttcgat
     2821 tgggaggaag tttgcctcac cagcgcttac aaggagctgt tcaataagta tgggattaac
     2881 taccagcagg gcgacataag agccctgctg tgcgaacaat ctgataaggc attctattcc
     2941 tctttcatgg cactgatgtc actgatgctg caaatgcgca attccatcac cggaagaaca
     3001 gacgtggcct ttctgatctc tcctgtcaag aactcagatg gcatcttcta cgattcccgc
     3061 aactatgaag cacaggagaa tgctatcctg cctaagaatg ccgatgcaaa tggagcctat
     3121 aacatcgcca gaaaggtcct ctgggccata ggacaattca agaaagctga agatgagaag
     3181 ctggacaagg tgaagatcgc catttcaaac aaagagtggc tcgaatatgc tcagacctca
     3241 gtgaagcatg gatcatctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc
     3301 aagaagaaga ggaaagtcgg ctcgggccag tgtactaatt atgctctctt gaaattggct
     3361 ggagatgttg agagcaaccc aggtcccgtg agcaagggcg aggagctgtt caccggggtg
     3421 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3481 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3541 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     3601 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3661 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3721 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3781 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3841 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3901 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3961 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     4021 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4081 ggcatggacg agctgtacaa gtaaatcatc accatcacca ttgagtttaa acccgctgat
     4141 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4201 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4261 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4321 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     4381 aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat
     4441 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4501 gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa
     4561 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4621 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4681 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4741 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4801 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4861 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4921 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4981 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5041 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5101 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5161 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5221 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5281 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5341 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5401 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5461 ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5521 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5581 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5641 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5701 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5761 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5821 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5881 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5941 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6001 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6061 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6121 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6181 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6241 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6301 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6361 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6421 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6481 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6541 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6601 tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct
     6661 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6721 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6781 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     6841 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     6901 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6961 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     7021 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     7081 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     7141 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     7201 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     7261 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     7321 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     7381 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     7441 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
     7501 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtctctgag
     7561 gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa gagggcacgc
     7621 gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acaatagagt gatcggcgag
     7681 ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat tatggccctg
     7741 agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct gtacgtgaca
     7801 ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatcgg ccgcgtggtg
     7861 tttggctgga ggaactcaaa aagaggcgcc gcaggctccc tgatgaacgt gctgaactac
     7921 cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga atgtgccgcc
     7981 ctgctgtgcg atttctatcg gatgcctaga caggtgttca atgctcagaa gaaggcccag
     8041 agctccatca actccggagg atctagcgga ggctcctctg gctctgagac acctggcaca
     8101 agcgagagcg caacacctga aagcagcggg ggcagcagcg gggggtcatc aaagctggag
     8161 aaattcacca actgttatag cctgtctaag accctgcgct tcaaggcaat cccagtgggc
     8221 aagacacaag agaacattga caacaaacgg ctcctggtgg aggatgagaa gagggctgaa
     8281 gattacaagg gcgttaagaa gctgctggat aggtactatc tgtcattcat caacgatgtc
     8341 ctccacagta tcaagctgaa gaatctgaac aattacattt ctctgttccg gaagaagaca
     8401 cggaccgaga aggagaacaa agagctggag aatctggaga tcaacctgag gaaagaaata
     8461 gctaaggctt tcaaagggaa cgagggttac aagtccctgt tcaagaaaga cattatcgag
     8521 actattctgc ctgagttcct ggacgataaa gatgagatcg ccctcgtcaa ttccttcaat
//
