LOCUS       sequence-237432-        5148 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 5148)
  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 5148)
  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..5148
                     /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"
     polyA_signal    1197..1331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1221..1240)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1275..1294
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1444..1464)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1444..1462)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1483..1499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1483..1499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1496..1518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1507..1523
                     /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(1531..1537,1538..1555,1556..1561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1576..1597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1714..1731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1885..2473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1965..1984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2644..3435,3436..3504))
                     /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     3267..3286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3505..3609)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3635..4090)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3772..3793)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3984..4003
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4164..5148
                     /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 ccttaagaat tcaaggcctc tcgagcctct agaactatag tgagtcgtat tacgtagatc
     1201 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     1261 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     1321 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     1381 gggaggtttt ttaattcgcg gccgcggcgc caatgcattg ggcccggtac ccagcttttg
     1441 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     1501 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     1561 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     1621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1681 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     1741 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     1801 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1861 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1921 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1981 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2041 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2101 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2161 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2221 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2281 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     2341 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2401 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2581 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2641 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2701 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2761 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2821 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2881 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2941 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3001 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3061 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3121 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3181 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3241 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3301 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3361 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3421 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3481 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3541 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3601 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     3661 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3721 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3781 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3841 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     3901 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3961 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4021 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4081 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4141 gcctcttcgc tattacgcca gtcgaccata gccaattcaa tatggcgtat atggactcat
     4201 gccaattcaa tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt
     4261 gccaattcaa tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat
     4321 gccaattcaa tatggcggac ttggcactgt gccaactggg gaggggtcta cttggcacgg
     4381 tgccaagttt gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg
     4441 tgccaataat ggcggccata ttggctatat gccaggatca atatataggc aatatccaat
     4501 atggccctat gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca
     4561 tatagtattc catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc
     4621 ccatatacca tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa
     4681 tggtctctat atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg
     4741 gcgcctcccc cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac
     4801 gtcaatagga ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt
     4861 tctcacgccc cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat
     4921 ctattaatag taacttggca agtacattac tattggaagg acgccagggt acattggcag
     4981 tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat
     5041 tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt
     5101 aggcgtgcct aatgggaggt ctatataagc aatgctcgtt tagggaac
//
