LOCUS       sequence-237558-        7177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipYFP (TEV cleavage sequence) and T2A mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7177)
  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 7177)
  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-FlipYFP(TEV cleavage seq) T2A mCherry
FEATURES             Location/Qualifiers
     source          1..7177
                     /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             1168..1188
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1297..1308
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1300..1353
                     /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             1969..2022
                     /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             2029..2739
                     /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(2174..2192)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2442..2461
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2771..2788)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2777..3001
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3047..3475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3134..3153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3344..3365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3484..3504)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3489..3818
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3669..3804
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3731..3750
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3885..4679
                     /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(3939..3958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4549..4568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4853..4974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4890..4909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4944..4963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5023..5039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5023..5039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5036..5058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5047..5063
                     /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(5071..5077,5078..5095,5096..5101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5116..5137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5254..5271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5425..6010)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5505..5524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6181..6972,6973..7041))
                     /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     6804..6823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7042..7146)
                     /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 ctgtcctacc 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 tgcatcagaa aacctgtact ttcagtccaa ggtgtccgcc
     1201 ctgaaggaaa aagtaagcgc actgaaagaa aaggtgagcg cgctgaagga gaaagtgagc
     1261 gccctgaaag agaaagtctc tgcccttaag gaggatatcg agggcagagg aagtctgcta
     1321 acatgcggtg acgtcgagga gaatcctggc ccaaagcttg ccaccatgcg caaaggcgaa
     1381 gaactgttta ccggcattgt gccgattctg gtggaactgg atggcgatgt gaacggccat
     1441 aaattttttg tgcgcggcga aggcgaaggc gatgcgacca ttggcaaact gagcctgaaa
     1501 tttatttgca ccaccggcaa actgccggtg ccgtggccga ccctggtgac caccctgacc
     1561 tatggcgtgc agtgctttag ccgctatccg gatcatatga aacgccatga tttttttaaa
     1621 agcgcgatgc cggaaggcta tgtgcaggaa cgcaccattt attttaaaga tgatggcacc
     1681 tataaaaccc gcgcggaagt gaaatttgaa ggcgataccc tggtgaaccg cattgaactg
     1741 aaaggcattg attttaaaga agatggcaac attctgggcc ataaactgga atataacttt
     1801 aacagccata aagtgtatat taccgcggat aaacagaaca acggcattaa agcgaacttt
     1861 accattcgcc ataacgtgga agatggcagc gtgcagctgg cggatcatta tcagcagaac
     1921 accccgattg gcgatggccc ggttcttctt cctggcggcc gctctagaga gggcagagga
     1981 agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagaattgat ggtgagcaag
     2041 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     2101 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     2161 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     2221 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     2281 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2341 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2401 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     2461 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2521 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2581 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2641 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2701 cgccactcca ccggcggcat ggacgagctg tacaagtaat ctagagggcc cgtttaaacc
     2761 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2821 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2881 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2941 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3001 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3061 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3301 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3361 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3421 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3481 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3541 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3601 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3661 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3721 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3781 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3841 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3901 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3961 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4021 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4081 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4141 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4201 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4261 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4321 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4381 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4441 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4501 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4561 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4621 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4681 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4741 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4801 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4861 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4921 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4981 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5041 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5101 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5161 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5221 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5281 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5341 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5401 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5461 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5521 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5581 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5641 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5701 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5761 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5821 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5881 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5941 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6001 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6061 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6121 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6181 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6241 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6301 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6361 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6421 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6481 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6541 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6601 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6661 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6721 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6781 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6841 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6901 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6961 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7021 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7081 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7141 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
