LOCUS       sequence-322471-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipGFP (caspase-3 cleavage sequence) and T2A mCherry in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  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 7186)
  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-FlipGFP(Casp3 cleavage seq) T2A mCherry
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1069..1080
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1072..1125
                     /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             1741..1794
                     /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             1801..2511
                     /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(1946..1964)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2214..2233
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2543..2560)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2549..2773
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2819..3247
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2906..2925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3116..3137
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3256..3276)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3261..3590
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3441..3576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3503..3522
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3657..4451
                     /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(3711..3730)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4321..4340
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4625..4746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4662..4681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4716..4735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4795..4811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4795..4811)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4808..4830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4819..4835
                     /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(4843..4849,4850..4867,4868..4873))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4888..4909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5026..5043)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5197..5785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5277..5296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5956..6747,6748..6816))
                     /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     6579..6598
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6817..6921)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6996..7015)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatggacct gcctgacgac cactacctgt ccacccagac catcctgtcc
      721 aaggacctga actccggact cagatctggc agcggtctcg agatggaagt tagcgctctg
      781 gaaaaagaag tgtctgcact cgagaaagaa gtaagtgccc ttgagaagga ggtgtccgca
      841 ctcgagaagg aggtcagcgc cctggaaaag gaaaagcgag accatatggt tttgcttgag
      901 tatgttacag cggctggcat taccgatgca tcaggtgatg aagttgatgg tggcggtggc
      961 aaggtgtccg ccctgaagga aaaagtaagc gcactgaaag aaaaggtgag cgcgctgaag
     1021 gagaaagtga gcgccctgaa agagaaagtc tctgccctta aggaggatat cgagggcaga
     1081 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaaagct tgccaccatg
     1141 cgcaaaggcg aagaactgtt taccggcatt gtgccgattc tggtggaact ggatggcgat
     1201 gtgaacggcc ataaattttt tgtgcgcggc gaaggcgaag gcgatgcgac cattggcaaa
     1261 ctgagcctga aatttatttg caccaccggc aaactgccgg tgccgtggcc gaccctggtg
     1321 accaccctga cctatggcgt gcagtgcttt agccgctatc cggatcatat gaaacgccat
     1381 gattttttta aaagcgcgat gccggaaggc tatgtgcagg aacgcaccat ttattttaaa
     1441 gatgatggca cctataaaac ccgcgcggaa gtgaaatttg aaggcgatac cctggtgaac
     1501 cgcattgaac tgaaaggcat tgattttaaa gaagatggca acattctggg ccataaactg
     1561 gaatataact ttaacagcca taaagtgtat attaccgcgg ataaacagaa caacggcatt
     1621 aaagcgaact ttaccattcg ccataacgtg gaagatggca gcgtgcagct ggcggatcat
     1681 tatcagcaga acaccccgat tggcgatggc ccggttcttc ttcctggcgg ccgctctaga
     1741 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagaattg
     1801 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     1861 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1921 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1981 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     2041 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     2101 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     2161 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     2221 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     2281 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     2341 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     2401 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     2461 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta atctagaggg
     2521 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2581 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2641 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2701 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2761 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     2821 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2881 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2941 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3001 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3061 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3121 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3181 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3241 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3301 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3361 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3421 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3481 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3541 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3601 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3661 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3721 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3781 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3841 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3901 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3961 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4021 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4081 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4141 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4201 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4261 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4321 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4381 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4441 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4501 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4561 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4621 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4681 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4741 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4801 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4861 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4921 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4981 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5041 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5101 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5161 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5221 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5281 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5341 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5401 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5461 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5521 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5581 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5641 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5701 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5761 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5821 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5881 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5941 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6001 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6061 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6121 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6181 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6241 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6301 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6361 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6421 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6481 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6541 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6601 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6661 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6721 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6781 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6841 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6901 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     6961 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     7021 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     7081 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     7141 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
