LOCUS       sequence-237434-        6734 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses GFP1-9 and T2A mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6734)
  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 6734)
  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-GFP1-9 T2A mCherry
FEATURES             Location/Qualifiers
     source          1..6734
                     /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             1526..1579
                     /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             1586..2296
                     /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(1731..1749)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1999..2018
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2328..2345)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2334..2558
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2604..3032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2691..2710)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2901..2922
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3041..3061)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3046..3375
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3226..3361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3288..3307
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3442..4236
                     /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(3496..3515)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4106..4125
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4410..4531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4447..4466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4501..4520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4580..4596)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4580..4596)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4593..4615)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4604..4620
                     /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(4628..4634,4635..4652,4653..4658))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4673..4694
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4811..4828)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4982..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5062..5081)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5738..6529,6530..6598))
                     /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     6361..6380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6599..6703)
                     /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 gtttaaactt aagcttgcca ccatgcgcaa aggcgaagaa ctgtttaccg gcattgtgcc
      961 gattctggtg gaactggatg gcgatgtgaa cggccataaa ttttttgtgc gcggcgaagg
     1021 cgaaggcgat gcgaccattg gcaaactgag cctgaaattt atttgcacca ccggcaaact
     1081 gccggtgccg tggccgaccc tggtgaccac cctgacctat ggcgtgcagt gctttagccg
     1141 ctatccggat catatgaaac gccatgattt ttttaaaagc gcgatgccgg aaggctatgt
     1201 gcaggaacgc accatttatt ttaaagatga tggcacctat aaaacccgcg cggaagtgaa
     1261 atttgaaggc gataccctgg tgaaccgcat tgaactgaaa ggcattgatt ttaaagaaga
     1321 tggcaacatt ctgggccata aactggaata taactttaac agccataaag tgtatattac
     1381 cgcggataaa cagaacaacg gcattaaagc gaactttacc attcgccata acgtggaaga
     1441 tggcagcgtg cagctggcgg atcattatca gcagaacacc ccgattggcg atggcccggt
     1501 tcttcttcct ggcggccgct ctagagaggg cagaggaagt ctgctaacat gcggtgacgt
     1561 cgaggagaat cctggcccag aattgatggt gagcaagggc gaggaggata acatggccat
     1621 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     1681 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     1741 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     1801 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     1861 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     1921 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     1981 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     2041 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     2101 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     2161 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     2221 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     2281 cgagctgtac aagtaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2341 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2401 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2461 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2521 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2581 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2641 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2701 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2761 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2821 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2881 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2941 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3001 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3121 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3181 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3241 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3301 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3361 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3421 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3481 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3541 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3601 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3661 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3721 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3781 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3841 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3901 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3961 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4021 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4081 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4141 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4201 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4261 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4321 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4381 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4501 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4561 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4621 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4681 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4741 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4801 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4861 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4921 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4981 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5041 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5101 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5161 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5221 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5281 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5341 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5401 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5461 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6721 tgccacctga cgtc
//
