LOCUS       sequence-372946-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FlipGFP (TEV cleavage sequence) and T2A mCherry in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  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 7180)
  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-FlipGFP(TEV cleavage seq) T2A mCherry
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     202..222
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(231..659)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(341..362)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     553..572
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    705..929
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     918..935
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(967..1677)
                     /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(1245..1264)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1514..1532
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1684..1737)
                     /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             complement(2353..2406)
                     /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             complement(2398..2409)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2518..2538)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      2796..2805
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2824..2843)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2825..2843)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2888..3091)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2917..2937)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3092..3471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3643..3662
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3737..3841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3842..3910,3911..4702)
                     /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     complement(4060..4079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4873..5461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5362..5381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5615..5632
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5749..5770
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5785..5790,5791..5808,5809..5815)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5823..5839
                     /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)."
     primer_bind     5828..5850
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5847..5863
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5847..5863
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5912..6033
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5923..5942)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5977..5996
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6207..7001)
                     /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(6318..6337)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6928..6947
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(7068..7180,1..217))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7082..7180,1..37)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7136..7155)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
       61 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
      121 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
      181 gggagcctgg ggactttcca caccctaact gacacacatt ccacagaatt aattcgcgtt
      241 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
      301 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
      361 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
      421 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
      481 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
      541 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
      601 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg
      661 gggatacccc ctagagcccc agctggttct ttccgcctca gaagccatag agcccaccgc
      721 atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc
      781 accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta
      841 ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac
      901 aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt aaacgggccc
      961 tctagattac ttgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgttc
     1021 gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac
     1081 gttgtaggcg ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc
     1141 gtcgtagtgg ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc
     1201 gtcctcgggg tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac
     1261 ggggccgtcg gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc
     1321 ctgcagggag gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg
     1381 ctcccacttg aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg
     1441 cttcacgtag gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg
     1501 cagggggcca cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc
     1561 ctcgccctcg ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt
     1621 gaagcgcatg aactccttga tgatggccat gttatcctcc tcgcccttgc tcaccatcaa
     1681 ttctgggcca ggattctcct cgacgtcacc gcatgttagc agacttcctc tgccctctct
     1741 agagcggccg ccaggaagaa gaaccgggcc atcgccaatc ggggtgttct gctgataatg
     1801 atccgccagc tgcacgctgc catcttccac gttatggcga atggtaaagt tcgctttaat
     1861 gccgttgttc tgtttatccg cggtaatata cactttatgg ctgttaaagt tatattccag
     1921 tttatggccc agaatgttgc catcttcttt aaaatcaatg cctttcagtt caatgcggtt
     1981 caccagggta tcgccttcaa atttcacttc cgcgcgggtt ttataggtgc catcatcttt
     2041 aaaataaatg gtgcgttcct gcacatagcc ttccggcatc gcgcttttaa aaaaatcatg
     2101 gcgtttcata tgatccggat agcggctaaa gcactgcacg ccataggtca gggtggtcac
     2161 cagggtcggc cacggcaccg gcagtttgcc ggtggtgcaa ataaatttca ggctcagttt
     2221 gccaatggtc gcatcgcctt cgccttcgcc gcgcacaaaa aatttatggc cgttcacatc
     2281 gccatccagt tccaccagaa tcggcacaat gccggtaaac agttcttcgc ctttgcgcat
     2341 ggtggcaagc tttgggccag gattctcctc gacgtcaccg catgttagca gacttcctct
     2401 gccctcgata tcctccttaa gggcagagac tttctctttc agggcgctca ctttctcctt
     2461 cagcgcgctc accttttctt tcagtgcgct tactttttcc ttcagggcgg acaccttgga
     2521 ctgaaagtac aggttttctg atgcatcggt aatgccagcc gctgtaacat actcaagcaa
     2581 aaccatatgg tctcgctttt ccttttccag ggcgctgacc tccttctcga gtgcggacac
     2641 ctccttctca agggcactta cttctttctc gagtgcagac acttcttttt ccagagcgct
     2701 aacttccatc tcgagaccgc tgccagatct gagtccggag ttcaggtcct tggacaggat
     2761 ggtctgggtg gacaggtagt ggtcgtcagg caggtccatg gtggcgctag ccagcttggg
     2821 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     2881 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     2941 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     3001 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     3061 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     3121 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     3181 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     3241 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     3301 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     3361 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     3421 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     3481 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     3541 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     3601 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     3661 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     3721 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3781 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3841 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3901 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3961 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4021 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4081 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4141 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4201 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4261 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4321 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4381 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4441 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4501 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4561 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4621 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4681 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4741 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4801 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4861 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4921 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4981 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5041 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5101 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5161 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5221 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5281 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5341 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5401 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5461 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5521 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5581 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5641 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5701 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5761 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5821 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     5881 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     5941 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     6001 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     6061 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     6121 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     6181 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     6241 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     6301 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     6361 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     6421 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     6481 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     6541 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     6601 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     6661 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     6721 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     6781 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     6841 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     6901 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     6961 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     7021 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     7081 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     7141 gcctctgcat aaataaaaaa aattagtcag ccatggggcg
//
