LOCUS       sequence-242306-        5928 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 5928)
  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 5928)
  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..5928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1063..1072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1465..1476
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1468..1521
                     /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             2137..2190
                     /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             2197..2907
                     /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(2342..2360)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2610..2629
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2958..3092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2982..3001)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3036..3055
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3205..3225)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3205..3223)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3244..3260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3244..3260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3257..3279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3268..3284
                     /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(3292..3298,3299..3316,3317..3322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3337..3358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3475..3492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3646..4234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3726..3745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4405..5196,5197..5265))
                     /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     5028..5047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5266..5370)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5396..5851)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5533..5554)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5745..5764
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ccgccaccat ggacctgcct
     1081 gacgaccact acctgtccac ccagaccatc ctgtccaagg acctgaactc cggactcaga
     1141 tctggcagcg gtctcgagat ggaagttagc gctctggaaa aagaagtgtc tgcactcgag
     1201 aaagaagtaa gtgcccttga gaaggaggtg tccgcactcg agaaggaggt cagcgccctg
     1261 gaaaaggaaa agcgagacca tatggttttg cttgagtatg ttacagcggc tggcattacc
     1321 gatgcatcag gtgatgaagt tgatggtggc ggtggcaagg tgtccgccct gaaggaaaaa
     1381 gtaagcgcac tgaaagaaaa ggtgagcgcg ctgaaggaga aagtgagcgc cctgaaagag
     1441 aaagtctctg cccttaagga ggatatcgag ggcagaggaa gtctgctaac atgcggtgac
     1501 gtcgaggaga atcctggccc aaagcttgcc accatgcgca aaggcgaaga actgtttacc
     1561 ggcattgtgc cgattctggt ggaactggat ggcgatgtga acggccataa attttttgtg
     1621 cgcggcgaag gcgaaggcga tgcgaccatt ggcaaactga gcctgaaatt tatttgcacc
     1681 accggcaaac tgccggtgcc gtggccgacc ctggtgacca ccctgaccta tggcgtgcag
     1741 tgctttagcc gctatccgga tcatatgaaa cgccatgatt tttttaaaag cgcgatgccg
     1801 gaaggctatg tgcaggaacg caccatttat tttaaagatg atggcaccta taaaacccgc
     1861 gcggaagtga aatttgaagg cgataccctg gtgaaccgca ttgaactgaa aggcattgat
     1921 tttaaagaag atggcaacat tctgggccat aaactggaat ataactttaa cagccataaa
     1981 gtgtatatta ccgcggataa acagaacaac ggcattaaag cgaactttac cattcgccat
     2041 aacgtggaag atggcagcgt gcagctggcg gatcattatc agcagaacac cccgattggc
     2101 gatggcccgg ttcttcttcc tggcggccgc tctagagagg gcagaggaag tctgctaaca
     2161 tgcggtgacg tcgaggagaa tcctggccca gaattgatgg tgagcaaggg cgaggaggat
     2221 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     2281 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2341 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     2401 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     2461 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2521 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     2581 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     2641 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     2701 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     2761 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     2821 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     2881 ggcggcatgg acgagctgta caagtaagaa ttcaaggcct ctcgagcctc tagaactata
     2941 gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca
     3001 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3061 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3121 ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt
     3181 gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     3241 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3301 agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3481 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3541 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3601 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3661 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3721 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3781 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3841 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3901 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3961 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4021 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4081 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4141 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4201 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4261 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4321 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4381 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4441 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4501 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4561 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4621 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4681 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4741 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4801 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4861 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4921 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4981 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5041 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5101 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5161 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5221 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5281 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5341 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     5401 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5461 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5521 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5581 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5641 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5701 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5761 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5821 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     5881 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agtcgacc
//
