LOCUS       sequence-224989-        6704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid for measuring protease activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6704)
  AUTHORS   Gao XJ, Chong LS, Kim MS, Elowitz MB
  TITLE     Programmable protein circuits in living cells.
  JOURNAL   Science. 2018 Sep 21;361(6408):1252-1258. doi: 
            10.1126/science.aat5062.
  PUBMED    30237357
REFERENCE   2  (bases 1 to 6704)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2018-09-21- 21"; volume: "361"; 
            issue: "6408"; pages: "1252-1258"
COMMENT     Sequence Label: pcDNA3.1-tvD-Cit-tevs-DHFR
FEATURES             Location/Qualifiers
     source          1..6704
                     /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      918..927
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1017..1019,1020..1730)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1059..1080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1320..1339)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1667..1688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1746..1766
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2295..2312)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2301..2525
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2571..2999
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2658..2677)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2868..2889
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3008..3028)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3013..3342
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3193..3328
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3255..3274
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3409..4203
                     /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(3463..3482)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4073..4092
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4377..4498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4414..4433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4468..4487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4547..4563)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4547..4563)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4560..4582)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4571..4587
                     /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(4595..4601,4602..4619,4620..4625))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4640..4661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4778..4795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4949..5537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5029..5048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5708..6499,6500..6568))
                     /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     6331..6350
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6569..6673)
                     /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 aagcttcgcc accatgggat ccggaggtag tgagacggtt agattccagt
      961 accacaaatc tggagcctgg aaacttccgg tgagtctggt aaagggcggt acctccgtga
     1021 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1081 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1141 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1201 ccaccttcgg ctacggcctg atgtgcttcg cccgctaccc cgaccacatg aagcagcacg
     1261 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1321 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1381 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1441 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1501 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1561 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1621 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1681 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggtgggtccg
     1741 gtctcgagaa cttgtacttc cagagtggga gcggatcgat atcactgatc gctgcactcg
     1801 cggttgatta tgtaataggc atggaaaacg cgatgccctg gaacctgcca gcagacctcg
     1861 catggtttaa gcggaacact ctgaataagc cagtcattat gggccgacat acgtgggaaa
     1921 gcattggacg cccactgcca ggaaggaaga atataatcct gtcatcacag ccgtccacgg
     1981 acgatagagt cacttgggta aaatccgttg acgaagccat agctgcctgt ggtgatgtgc
     2041 ctgagataat ggtaatagga ggagggcggg taatcgaaca atttctgcct aaagcccaga
     2101 agctgtacct tacccatatt gacgcggaag tagagggcga cacacatttc ccagattatg
     2161 agccagatga ttgggagagc gttttctccg aatttcatga tgcagacgcc cagaatagcc
     2221 acagctactg ctttgaaata ttggagcgcc gatgaagcta gctcgaggag ggcccgttta
     2281 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2341 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2401 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2461 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2521 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2581 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2641 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2701 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2761 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2821 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2881 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2941 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3001 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3061 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3121 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3181 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3241 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     3301 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     3361 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3421 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3481 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3541 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     3601 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3661 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3721 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3781 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3841 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3901 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     3961 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4021 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4081 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4141 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4201 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     4261 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4321 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4381 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4441 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4501 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4561 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4621 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4681 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4741 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4801 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4861 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4921 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4981 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5041 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5101 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5161 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5221 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5281 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5341 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5401 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5461 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5521 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5581 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5641 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5701 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5761 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5821 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5881 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5941 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6001 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6061 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6121 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6181 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6241 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6301 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6361 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6421 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6481 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6541 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6601 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6661 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
