LOCUS       sequence-237433-        5931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipGFP (caspase-3 cleavage sequence) protease reporter 
            and T2A mCherry in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5931)
  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 5931)
  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: PCS2-FlipGFP(Casp3 cleavage seq) T2A mCherry
FEATURES             Location/Qualifiers
     source          1..5931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             487..498
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             490..543
                     /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             1159..1212
                     /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             1219..1929
                     /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(1364..1382)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1632..1651
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1980..2114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2004..2023)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2058..2077
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2227..2247)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2227..2245)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2266..2282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2266..2282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2279..2301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2290..2306
                     /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(2314..2320,2321..2338,2339..2344))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2359..2380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2497..2514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2668..3256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2748..2767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3427..4218,4219..4287))
                     /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     4050..4069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4288..4392)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4418..4873)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4555..4576)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4767..4786
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4947..5931
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccgccacc atggacctgc ctgacgacca ctacctgtcc
      121 acccagacca tcctgtccaa ggacctgaac tccggactca gatctggcag cggtctcgag
      181 atggaagtta gcgctctgga aaaagaagtg tctgcactcg agaaagaagt aagtgccctt
      241 gagaaggagg tgtccgcact cgagaaggag gtcagcgccc tggaaaagga aaagcgagac
      301 catatggttt tgcttgagta tgttacagcg gctggcatta ccgatgcatc aggtgatgaa
      361 gttgatggtg gcggtggcaa ggtgtccgcc ctgaaggaaa aagtaagcgc actgaaagaa
      421 aaggtgagcg cgctgaagga gaaagtgagc gccctgaaag agaaagtctc tgcccttaag
      481 gaggatatcg agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
      541 ccaaagcttg ccaccatgcg caaaggcgaa gaactgttta ccggcattgt gccgattctg
      601 gtggaactgg atggcgatgt gaacggccat aaattttttg tgcgcggcga aggcgaaggc
      661 gatgcgacca ttggcaaact gagcctgaaa tttatttgca ccaccggcaa actgccggtg
      721 ccgtggccga ccctggtgac caccctgacc tatggcgtgc agtgctttag ccgctatccg
      781 gatcatatga aacgccatga tttttttaaa agcgcgatgc cggaaggcta tgtgcaggaa
      841 cgcaccattt attttaaaga tgatggcacc tataaaaccc gcgcggaagt gaaatttgaa
      901 ggcgataccc tggtgaaccg cattgaactg aaaggcattg attttaaaga agatggcaac
      961 attctgggcc ataaactgga atataacttt aacagccata aagtgtatat taccgcggat
     1021 aaacagaaca acggcattaa agcgaacttt accattcgcc ataacgtgga agatggcagc
     1081 gtgcagctgg cggatcatta tcagcagaac accccgattg gcgatggccc ggttcttctt
     1141 cctggcggcc gctctagaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     1201 aatcctggcc cagaattgat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     1261 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     1321 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     1381 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     1441 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     1501 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     1561 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     1621 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     1681 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     1741 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     1801 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     1861 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     1921 tacaagtaag aattcaaggc ctctcgagcc tctagaacta tagtgagtcg tattacgtag
     1981 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2041 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2101 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2161 tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacccagctt
     2221 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2281 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2341 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2401 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2461 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2521 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2581 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2641 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2701 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2761 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2821 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2881 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2941 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3001 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3061 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3121 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3181 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3241 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3301 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3361 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3421 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3481 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3541 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3601 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3661 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3721 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3781 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3841 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3901 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3961 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4021 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4081 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4141 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4201 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4261 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4321 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4381 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4441 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4501 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4561 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4621 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4681 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4741 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4801 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4861 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     4921 cgggcctctt cgctattacg ccagtcgacc atagccaatt caatatggcg tatatggact
     4981 catgccaatt caatatggtg gatctggacc tgtgccaatt caatatggcg tatatggact
     5041 cgtgccaatt caatatggtg gatctggacc ccagccaatt caatatggcg gacttggcac
     5101 catgccaatt caatatggcg gacttggcac tgtgccaact ggggaggggt ctacttggca
     5161 cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag tccgccatat tgaattggca
     5221 tggtgccaat aatggcggcc atattggcta tatgccagga tcaatatata ggcaatatcc
     5281 aatatggccc tatgccaata tggctattgg ccaggttcaa tactatgtat tggccctatg
     5341 ccatatagta ttccatatat gggttttcct attgacgtag atagcccctc ccaatgggcg
     5401 gtcccatata ccatatatgg ggcttcctaa taccgcccat agccactccc ccattgacgt
     5461 caatggtctc tatatatggt ctttcctatt gacgtcatat gggcggtcct attgacgtat
     5521 atggcgcctc ccccattgac gtcaattacg gtaaatggcc cgcctggctc aatgcccatt
     5581 gacgtcaata ggaccaccca ccattgacgt caatgggatg gctcattgcc cattcatatc
     5641 cgttctcacg ccccctattg acgtcaatga cggtaaatgg cccacttggc agtacatcaa
     5701 tatctattaa tagtaacttg gcaagtacat tactattgga aggacgccag ggtacattgg
     5761 cagtactccc attgacgtca atggcggtaa atggcccgcg atggctgcca agtacatccc
     5821 cattgacgtc aatggggagg ggcaatgacg caaatgggcg ttccattgac gtaaatgggc
     5881 ggtaggcgtg cctaatggga ggtctatata agcaatgctc gtttagggaa c
//
