LOCUS       sequence-450497-        4923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively expresses mCherry as a transfection marker. Expresses
            non-fluorescent EGFP, deactivated by A111V mutation, that can be 
            reactivated by active adenine base editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4923)
  AUTHORS   Osgood NRB, Zawalick NM, Sawyer CB, Cowan QT, Gu S, Mawson SJ, 
            Ranzau BL, Li L, Gymrek M, Goren A, Komor AC
  TITLE     Genome editing with programmable base editors in human cells
  JOURNAL   Methods in Enzymology, February 5, 2025
REFERENCE   2  (bases 1 to 4923)
  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: "Methods in 
            Enzymology, February 5, 2025"
COMMENT     Sequence Label: pABE-dGFP
FEATURES             Location/Qualifiers
     source          1..4923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        36..415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        416..619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     570..590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     661..680
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        661..679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      687..696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             693..1400
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(838..856)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1106..1125
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1407..1463
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1464..1466,1467..1469,1470..2180)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRVEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1509..1530)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1770..1789)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2117..2138
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2193..2213
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2222..2239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2262..2279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2268..2492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2563..2579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2563..2579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2576..2598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2587..2603
                     /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(2611..2617,2618..2635,2636..2641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2656..2677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2794..2811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2965..3553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3045..3064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3724..4515,4516..4584))
                     /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     4347..4366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4585..4689)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4768..4787)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
       61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      601 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc
      661 taatacgact cactataggg agagccgcca ccatggtgag caagggcgag gaggataaca
      721 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
      781 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
      841 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
      901 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
      961 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1021 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1081 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1141 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     1201 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     1261 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     1321 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     1381 gcatggacga gctgtacaag agcggagcta ctaacttcag cctgctgaag caggctggag
     1441 acgtggagga gaaccctgga cctatggtga gcaagggcga ggagctgttc accggggtgg
     1501 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1561 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1621 agctgcccgt gccctggccc accctcgtga ccaccctgac ctatggagtg cagtgcttca
     1681 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1741 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgtagagg
     1801 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1861 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1921 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1981 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2041 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2101 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2161 gcatggacga gctgtacaag tctggtggtt ctcccaagaa gaaaaggaaa gtctaaccgg
     2221 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     2281 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2341 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2401 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2461 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2521 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     2581 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2641 agcctaggat gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2701 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcgggaag
     2761 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2821 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2881 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2941 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3001 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3061 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3121 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3181 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3241 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3301 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3361 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3421 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3481 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3541 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga
     3601 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3661 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3721 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3781 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3841 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3901 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3961 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4021 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4081 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4141 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4201 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4261 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4321 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4381 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4441 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4501 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4561 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4621 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4681 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     4741 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     4801 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     4861 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     4921 tgc
//
