LOCUS       sequence-225385-        8300 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 8300)
  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 8300)
  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: PB-PGK-FKBP-Zte-Cit-tv-DHFR
FEATURES             Location/Qualifiers
     source          1..8300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1414..1913
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1449..1472)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1846..1865
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1932..1941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2382..2402
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2415..2417,2418..3128)
                     /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(2457..2478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2718..2737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3065..3086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3144..3164
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     primer_bind     complement(3680..3697)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3686..3893
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3926..3946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3931..4260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4111..4246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4173..4192
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4308..4355
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4374..4748
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(4808..4828)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4870..4991
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4907..4926)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4961..4980
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5754..5788
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6279..6295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6279..6295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6292..6314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6303..6319
                     /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(6327..6333,6334..6351,6352..6357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6372..6393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6510..6527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6681..7269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6761..6780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7440..8231,8232..8300))
                     /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     8063..8082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtaggcgttt tgcgctgctt
     1381 cgcgatgtac gggccagata tacgcgttct accgggtagg ggaggcgctt ttcccaaggc
     1441 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     1501 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     1561 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     1621 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcat tagtctcgtg cagatggaca
     1681 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     1741 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     1801 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     1861 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     1921 gcccaagctt cgccaccatg ggaggagttc aggtggagac catatctccg ggggatggca
     1981 ggacatttcc gaaaaggggc cagacgtgtg ttgttcatta tacagggatg ttggaggacg
     2041 gaaaaaaggt agactcttct cgggatagaa ataagccctt taagttcatg ctgggtaagc
     2101 aggaagttat tcgaggatgg gaagaaggag tagcccagat gtcagttgga cagcgggcaa
     2161 aattgactat aagccctgat tatgcctacg gtgcaacggg tcacccaggt attataccgc
     2221 cgcatgcgac actcgttttt gacgtggaac ttttgaaacc tgaaggaggc ggatccgagc
     2281 aactcgaaaa gaagctgcaa gcgttggaga agaagctcgc acaacttgaa tggaaaaatc
     2341 aggcactgga aaagaaactg gctcaaggtg gctccggcag cgagaatctt tatttccaat
     2401 ccggctcagg ctccgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2461 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     2521 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2581 cctggcccac cctcgtgacc accttcggct acggcctgat gtgcttcgcc cgctaccccg
     2641 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2701 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2761 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2821 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2881 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2941 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3001 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg
     3061 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     3121 tgtacaaggg tgggtccggt ctcgagacgg ttagattcca gagtgggagc ggatcgatat
     3181 cactgatcgc tgcactcgcg gttgattatg taataggcat ggaaaacgcg atgccctgga
     3241 acctgccagc agacctcgca tggtttaagc ggaacactct gaataagcca gtcattatgg
     3301 gccgacatac gtgggaaagc attggacgcc cactgccagg aaggaagaat ataatcctgt
     3361 catcacagcc gtccacggac gatagagtca cttgggtaaa atccgttgac gaagccatag
     3421 ctgcctgtgg tgatgtgcct gagataatgg taataggagg agggcgggta atcgaacaat
     3481 ttctgcctaa agcccagaag ctgtacctta cccatattga cgcggaagta gagggcgaca
     3541 cacatttccc agattatgag ccagatgatt gggagagcgt tttctccgaa tttcatgatg
     3601 cagacgccca gaatagccac agctactgct ttgaaatatt ggagcgccga tgaagctagc
     3661 gaattcgaat ttaaatcggc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3721 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3781 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3841 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggacgcggcc
     3901 gcaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3961 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4021 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4081 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4141 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4201 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4261 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat
     4321 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     4381 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     4441 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     4501 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     4561 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     4621 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     4681 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     4741 aggactgagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4801 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4861 cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4921 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4981 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc
     5041 atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc
     5101 caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca
     5161 caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat
     5221 tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc
     5281 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     5341 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     5401 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     5461 ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac
     5521 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt
     5581 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac
     5641 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat
     5701 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt
     5761 caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg
     5821 gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga
     5881 agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg
     5941 ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc
     6001 catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg
     6061 gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac
     6121 cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg
     6181 atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc
     6241 ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     6301 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6361 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6421 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6481 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6541 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6601 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6661 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6721 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6781 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6841 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6901 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6961 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7021 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7081 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7141 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7201 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7261 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7321 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7381 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7441 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7501 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7561 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7621 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7681 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7741 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7801 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7861 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7921 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7981 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     8041 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     8101 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     8161 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     8221 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     8281 cggaaatgtt gaatactcat
//
