LOCUS       sequence-242305-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP1-9 and T2A mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Zhang Q, Schepis A, Huang H, Yang J, Ma W, Torra J, Zhang SQ, Yang 
            L, Wu H, Nonell S, Dong Z, Kornberg TB, Coughlin SR, Shu X
  TITLE     Designing a green fluorogenic protease reporter by flipping a beta 
            strand of GFP for imaging apoptosis in animals.
  JOURNAL   J Am Chem Soc. 2019 Mar 1. doi: 10.1021/jacs.8b13042.
  PUBMED    30821975
REFERENCE   2  (bases 1 to 6737)
  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: "J Am Chem 
            Soc. 2019 Mar 1. doi: 10.1021/jacs.8b13042."
COMMENT     Sequence Label: PCDNA3-GFP1-9 T2A mCherry
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             912..965
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             972..1682
                     /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(1117..1135)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1385..1404
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1714..1731)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1720..1944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1990..2418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2077..2096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2287..2308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2427..2447)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2432..2761
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2612..2747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2674..2693
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2828..3622
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2882..2901)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3492..3511
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3796..3917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3833..3852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3887..3906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3966..3982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3966..3982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3979..4001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3990..4006
                     /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(4014..4020,4021..4038,4039..4044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4059..4080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4197..4214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4368..4956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4448..4467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5127..5918,5919..5987))
                     /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     5750..5769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5988..6092)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6167..6186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6358..6737
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
      301 ttgccaccat gcgcaaaggc gaagaactgt ttaccggcat tgtgccgatt ctggtggaac
      361 tggatggcga tgtgaacggc cataaatttt ttgtgcgcgg cgaaggcgaa ggcgatgcga
      421 ccattggcaa actgagcctg aaatttattt gcaccaccgg caaactgccg gtgccgtggc
      481 cgaccctggt gaccaccctg acctatggcg tgcagtgctt tagccgctat ccggatcata
      541 tgaaacgcca tgattttttt aaaagcgcga tgccggaagg ctatgtgcag gaacgcacca
      601 tttattttaa agatgatggc acctataaaa cccgcgcgga agtgaaattt gaaggcgata
      661 ccctggtgaa ccgcattgaa ctgaaaggca ttgattttaa agaagatggc aacattctgg
      721 gccataaact ggaatataac tttaacagcc ataaagtgta tattaccgcg gataaacaga
      781 acaacggcat taaagcgaac tttaccattc gccataacgt ggaagatggc agcgtgcagc
      841 tggcggatca ttatcagcag aacaccccga ttggcgatgg cccggttctt cttcctggcg
      901 gccgctctag agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg
      961 gcccagaatt gatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1021 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1081 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1141 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1201 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1261 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1321 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1381 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1441 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1501 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     1561 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1621 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     1681 aatctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     1741 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1801 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     1861 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     1921 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     1981 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2041 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2101 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2161 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2221 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2281 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2341 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2401 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2461 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2521 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2581 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2641 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2701 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2761 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     2821 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2881 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2941 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3001 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3061 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3121 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3181 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3241 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3301 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3361 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3421 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3481 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3541 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3601 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3661 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3721 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3781 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3841 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3901 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3961 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4021 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4081 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4141 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4201 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4261 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4321 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4381 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4441 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4501 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4561 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4621 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4681 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4741 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4801 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4861 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4921 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4981 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5041 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5101 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5161 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5221 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5281 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5341 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5401 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5461 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5521 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5581 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5641 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5701 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5761 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5821 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5881 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5941 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6001 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6061 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6121 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6181 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6241 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6301 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     6361 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     6421 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6481 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     6541 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     6601 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6661 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     6721 tcatcgctat taccatg
//
