LOCUS       sequence-237428-        7186 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses FlipCherry (TEV cleavage sequence) and T2A GFP in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7186)
  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 7186)
  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-FlipCherry-T2A-GFP
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     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"
     CDS             1180..1200
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1318..1329
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1321..1374
                     /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"
     primer_bind     complement(1520..1538)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     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             join(2029..2031,2032..2034,2035..2748)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2074..2095)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2335..2354)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2682..2703
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2780..2797)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2786..3010
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3056..3484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3143..3162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3353..3374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3493..3513)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3498..3827
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3678..3813
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3740..3759
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3894..4688
                     /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(3948..3967)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4558..4577
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4862..4983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4899..4918)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4953..4972
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5032..5048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5032..5048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5045..5067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5056..5072
                     /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(5080..5086,5087..5104,5105..5110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5125..5146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5263..5280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5434..6019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5514..5533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6190..6981,6982..7050))
                     /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     6813..6832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7051..7155)
                     /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 gccaccatgc agcttcccgg cgcctacaac gtggacatca agctggacat caccagccac
      961 aacgaggact ccggactcag atctggcagc ggtctcgaga tggaagttag cgctctggaa
     1021 aaagaagtgt ctgcactcga gaaagaagta agtgcccttg agaaggaggt gtccgcactc
     1081 gagaaggagg tcagcgccct ggaaaagggc ggcagcggcg actacaccat cgtggagcag
     1141 tacgagagag ccgaggccag acgcagcacc gacgcaagcg aaaacctgta ctttcagtcc
     1201 ggcggcggca aggtgtccgc cctgaaggaa aaagtaagcg cactgaaaga aaaggtgagc
     1261 gcgctgaagg agaaagtgag cgccctgaaa gagaaagtct ctgcccttaa ggaggatatc
     1321 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaaagctt
     1381 gccaccatga aggaggacaa catggccatc atcaaggagt tcatgagatt caaggtgcac
     1441 atggatggct gcgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccacccc
     1501 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc
     1561 tgggacatcc tgagccccca gttcatgtac ggcagcaagg cctacgtgaa gcaccccgcc
     1621 gacatccccg actacctgaa gctgagcttc cccgagggct tcacctggga gagagtgatg
     1681 aacttcgagg acggcggcgt ggtgaccgtg acccaggaca gcagcctgca ggacggcgag
     1741 ttcatctaca aggtgaagct gctgggcatc aacttcccca gcgacggccc cgtgatgcag
     1801 aagaagacca tgggctggga ggccagcacc gagagaatgt accccgagga cggcgccctg
     1861 aagggcgaga tcaaccagag actgaagctg aaggacggcg gccactacga cgccgaggtg
     1921 aagaccacct acaaggccaa gaagcccgtg cagggcggcc gctctagaga gggcagagga
     1981 agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagaattgat ggtgagcaag
     2041 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2101 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2161 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2221 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2281 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2341 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2401 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2461 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc
     2521 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2581 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2641 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2701 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaatc tagagggccc
     2761 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2821 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2881 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2941 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3001 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3061 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3121 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3181 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3241 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3301 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3361 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3421 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3481 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3541 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3601 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3661 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3721 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3781 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3841 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3901 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3961 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4021 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4081 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4141 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4201 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4261 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4321 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4381 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4441 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4501 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4561 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4621 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4681 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4741 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4801 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4861 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4921 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4981 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5161 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5221 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5281 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5341 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5401 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5461 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5521 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5581 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5641 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5701 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5761 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5821 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5881 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5941 atccggcaaa caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc agattacgcg
     6001 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6061 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6121 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6181 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6301 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7141 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
