LOCUS       sequence-242300-        5145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipGFP (caspase-3 cleavage sequence) protease reporter in
            zebrafish (verified).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5145)
  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 5145)
  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)
FEATURES             Location/Qualifiers
     source          1..5145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        159..1143
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1178..1196
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1178..1195
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1228..1237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1630..1641
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1633..1686
                     /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"
     polyA_signal    2340..2474
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2364..2383)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2418..2437
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2587..2607)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2587..2605)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2626..2642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2626..2642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2639..2661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2650..2666
                     /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(2674..2680,2681..2698,2699..2704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2719..2740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2857..2874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3028..3616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3108..3127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3787..4578,4579..4647))
                     /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     4410..4429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4648..4752)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4778..5145,1..88))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4915..4936)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5127..5145,1)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
       61 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
      121 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa ttcaatatgg
      181 cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa ttcaatatgg
      241 cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa ttcaatatgg
      301 cggacttggc accatgccaa ttcaatatgg cggacctggc actgtgccaa ctggggaggg
      361 gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca agtccgccat
      421 attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag gatcaatata
      481 taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc aatactatgt
      541 attggcccta tgccatatag tattccatat atgggttttc ctattgacgt agatagcccc
      601 tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc atagccactc
      661 ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat atgggcggtc
      721 ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg cccgcctggc
      781 tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga tggctcattg
      841 cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat ggcccacttg
      901 gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg gaagtacgcc
      961 agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg cgatggctgc
     1021 caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg cgttccattg
     1081 acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc tcgtttaggg
     1141 aaccgccatt ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac
     1201 aagctacttg ttctttttgc aggatccgcc accatggacc tgcctgacga ccactacctg
     1261 tccacccaga ccatcctgtc caaggacctg aactccggac tcagatctgg cagcggtctc
     1321 gagatggaag ttagcgctct ggaaaaagaa gtgtctgcac tcgagaaaga agtaagtgcc
     1381 cttgagaagg aggtgtccgc actcgagaag gaggtcagcg ccctggaaaa ggaaaagcga
     1441 gaccatatgg ttttgcttga gtatgttaca gcggctggca ttaccgatgc atcaggtgat
     1501 gaagttgatg gtggcggtgg caaggtgtcc gccctgaagg aaaaagtaag cgcactgaaa
     1561 gaaaaggtga gcgcgctgaa ggagaaagtg agcgccctga aagagaaagt ctctgccctt
     1621 aaggaggata tcgagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     1681 ggcccaaagc ttgccaccat gcgcaaaggc gaagaactgt ttaccggcat tgtgccgatt
     1741 ctggtggaac tggatggcga tgtgaacggc cataaatttt ttgtgcgcgg cgaaggcgaa
     1801 ggcgatgcga ccattggcaa actgagcctg aaatttattt gcaccaccgg caaactgccg
     1861 gtgccgtggc cgaccctggt gaccaccctg acctatggcg tgcagtgctt tagccgctat
     1921 ccggatcata tgaaacgcca tgattttttt aaaagcgcga tgccggaagg ctatgtgcag
     1981 gaacgcacca tttattttaa agatgatggc acctataaaa cccgcgcgga agtgaaattt
     2041 gaaggcgata ccctggtgaa ccgcattgaa ctgaaaggca ttgattttaa agaagatggc
     2101 aacattctgg gccataaact ggaatataac tttaacagcc ataaagtgta tattaccgcg
     2161 gataaacaga acaacggcat taaagcgaac tttaccattc gccataacgt ggaagatggc
     2221 agcgtgcagc tggcggatca ttatcagcag aacaccccga ttggcgatgg cccggttctt
     2281 cttccttaag aattcaaggc ctctcgagcc tctagaacta tagtgagtcg tattacgtag
     2341 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2401 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2461 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2521 tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacccagctt
     2581 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2641 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     2701 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2761 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2821 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2881 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2941 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3001 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3061 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3121 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3181 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3241 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3301 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3361 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3421 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3481 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3541 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3601 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3661 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3721 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3781 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3841 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3901 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3961 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4021 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4081 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4141 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4201 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4261 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4321 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4381 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4441 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4501 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4561 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4621 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4681 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4741 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4801 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4861 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4921 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4981 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5041 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5101 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aagga
//
