LOCUS       sequence-198274-        7003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET-based ECFP-YPET reporter that is cleaved by TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7003)
  AUTHORS   Gray DC, Mahrus S, Wells JA
  TITLE     Activation of specific apoptotic caspases with an engineered 
            small-molecule-activated protease.
  JOURNAL   Cell. 2010 Aug 20;142(4):637-46. doi: 10.1016/j.cell.2010.07.014.
  PUBMED    20723762
REFERENCE   2  (bases 1 to 7003)
  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"; doi: 
            "10.1016/j.cell.2010.07"; journalName: "Cell"; date: "2010-08-20- 
            20"; volume: "142"; issue: "4"; pages: "637-46"
COMMENT     Sequence Label: ECFP-TevS-YPET
FEATURES             Location/Qualifiers
     source          1..7003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(730..732,733..1443)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     1380..1401
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1459..1479
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1495..1497,1498..2208)
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLE
                     FLTAAGITEGMNELYK"
     CDS             2209..2250
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2273..2302
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2318..2335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2358..2375)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2364..2588
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2634..3062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2721..2740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2931..2952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3071..3091)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3076..3405
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3256..3391
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3318..3337
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3472..4266
                     /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(3526..3545)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4136..4155
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4442..4563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4479..4498)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4533..4552
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4612..4628)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4612..4628)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4625..4647)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4636..4652
                     /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(4660..4666,4667..4684,4685..4690))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4705..4726
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4843..4860)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5014..5602)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5094..5113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5773..6564,6565..6633))
                     /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     6396..6415
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6634..6738)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6813..6832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag cttggtaccg agctcggatc tccaccatgg gaggctccct cgagggaggc
      721 tccggatccg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      781 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      841 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      901 cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac
      961 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1021 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1081 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1141 gggcacaagc tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag
     1201 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1261 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1321 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1381 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgaactgtac
     1441 aaggggggct ccggctccga aaatctctac ttccagagtg gcagcggagg ctccgtgagc
     1501 aaaggcgaag agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
     1561 aacggccaca agttcagcgt gagcggcgag ggcgagggcg acgccaccta cggcaagctg
     1621 accctgaagc tgctgtgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
     1681 accctgggct acggcgtgca gtgcttcgcc cggtaccccg accacatgaa gcagcacgac
     1741 ttcttcaaga gcgccatgcc cgagggctac gtgcaggagc ggaccatctt cttcaaggac
     1801 gacggcaact acaagacccg ggccgaggtg aagttcgagg gcgacaccct ggtgaaccgg
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctgggcca caagctggag
     1921 tacaactaca acagccacaa cgtgtacatc accgccgaca agcagaagaa cggcatcaag
     1981 gccaacttca agatccggca caacatcgag gacggcggcg tgcagctggc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 taccagagcg ccctgttcaa ggaccccaac gagaagcggg accacatggt gctgctggag
     2161 ttcctgaccg ccgccggcat caccgagggc atgaacgagc tctataaggg taagcctatc
     2221 cctaaccctc tcctcggtct cgattctacg taaactagag ggcccgcggt tcgaacaaaa
     2281 actcatctca gaagaggatc tgaatatgca taccggtcat catcaccatc accattgagt
     2341 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2401 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2461 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2521 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2581 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2641 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2701 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2761 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2821 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2881 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2941 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3001 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3061 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3121 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3181 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3241 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3301 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3361 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3421 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3481 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3541 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3601 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3661 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3721 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3781 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3841 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3901 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3961 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4021 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4081 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4141 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4201 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4261 ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc
     4321 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4381 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4441 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4501 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4561 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4621 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4681 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4741 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4801 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4861 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4921 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4981 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5041 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5101 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5161 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5221 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5281 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5341 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5401 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5461 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5521 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5581 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5641 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5701 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5761 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5821 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5881 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5941 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6001 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6061 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6121 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6181 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6241 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6301 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6361 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6421 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6481 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6541 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6601 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6661 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6721 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     6781 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6841 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     6901 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     6961 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
