LOCUS       sequence-243079-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipGFP (caspase-1 cleavage sequence) and T2A mCherry.
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(Caspase-1 cleavage seq) T2A mCh...
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1303..1314
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1306..1359
                     /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             1975..2028
                     /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             2035..2745
                     /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(2180..2198)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2448..2467
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2777..2794)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2783..3007
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3053..3481
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3140..3159)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3350..3371
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3490..3510)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3495..3824
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3675..3810
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3737..3756
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3891..4685
                     /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(3945..3964)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4555..4574
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4859..4980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4896..4915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4950..4969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5029..5045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5029..5045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5042..5064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5053..5069
                     /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(5077..5083,5084..5101,5102..5107))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5122..5143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5260..5277)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5431..6019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5511..5530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6190..6981,6982..7050))
                     /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     6813..6832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7051..7155)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg acctgcctga cgaccactac ctgtccaccc agaccatcct gtccaaggac
      961 ctgaactccg gactcagatc tggcagcggt ctcgagatgg aagttagcgc tctggaaaaa
     1021 gaagtgtctg cactcgagaa agaagtaagt gcccttgaga aggaggtgtc cgcactcgag
     1081 aaggaggtca gcgccctgga aaaggaaaag cgagaccata tggttttgct tgagtatgtt
     1141 acagcggctg gcattaccga tgcatcaggt tgggagcacg atggtggcgg tggcaaggtg
     1201 tccgccctga aggaaaaagt aagcgcactg aaagaaaagg tgagcgcgct gaaggagaaa
     1261 gtgagcgccc tgaaagagaa agtctctgcc cttaaggagg atatcgaggg cagaggaagt
     1321 ctgctaacat gcggtgacgt cgaggagaat cctggcccaa agcttgccac catgcgcaaa
     1381 ggcgaagaac tgtttaccgg cattgtgccg attctggtgg aactggatgg cgatgtgaac
     1441 ggccataaat tttttgtgcg cggcgaaggc gaaggcgatg cgaccattgg caaactgagc
     1501 ctgaaattta tttgcaccac cggcaaactg ccggtgccgt ggccgaccct ggtgaccacc
     1561 ctgacctatg gcgtgcagtg ctttagccgc tatccggatc atatgaaacg ccatgatttt
     1621 tttaaaagcg cgatgccgga aggctatgtg caggaacgca ccatttattt taaagatgat
     1681 ggcacctata aaacccgcgc ggaagtgaaa tttgaaggcg ataccctggt gaaccgcatt
     1741 gaactgaaag gcattgattt taaagaagat ggcaacattc tgggccataa actggaatat
     1801 aactttaaca gccataaagt gtatattacc gcggataaac agaacaacgg cattaaagcg
     1861 aactttacca ttcgccataa cgtggaagat ggcagcgtgc agctggcgga tcattatcag
     1921 cagaacaccc cgattggcga tggcccggtt cttcttcctg gcggccgctc tagagagggc
     1981 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaga attgatggtg
     2041 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     2101 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     2161 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     2221 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     2281 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     2341 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     2401 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     2461 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     2521 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     2581 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     2641 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     2701 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaatctag agggcccgtt
     2761 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2821 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2881 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2941 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3001 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     3061 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3121 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3181 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3241 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3301 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3361 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3421 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3481 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3541 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3601 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3661 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3721 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3781 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3841 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3901 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3961 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4021 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4081 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4141 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4201 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4261 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4321 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4381 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4441 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4501 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4561 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4621 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4681 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4741 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4801 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4861 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4921 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4981 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5041 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5101 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5161 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5221 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5281 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5341 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5401 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5461 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5521 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5581 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5641 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5701 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5761 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5821 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5881 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5941 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6001 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6061 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6121 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6181 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6301 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7141 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
