LOCUS       sequence-225376-        8309 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 8309)
  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 8309)
  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-hc-DHFR
FEATURES             Location/Qualifiers
     source          1..8309
                     /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"
     primer_bind     complement(3689..3706)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3695..3902
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3935..3955)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3940..4269
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4120..4255
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4182..4201
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4317..4364
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4383..4757
                     /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(4817..4837)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4879..5000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4916..4935)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4970..4989
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5763..5797
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6288..6304)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6288..6304)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6301..6323)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6312..6328
                     /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(6336..6342,6343..6360,6361..6366))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6381..6402
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6519..6536)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6690..7278)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6770..6789)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7449..8240,8241..8309))
                     /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     8072..8091
                     /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 ctcgacgaga tggaggagtg tagtcagcac cttgggagcg
     3181 gatcgatatc actgatcgct gcactcgcgg ttgattatgt aataggcatg gaaaacgcga
     3241 tgccctggaa cctgccagca gacctcgcat ggtttaagcg gaacactctg aataagccag
     3301 tcattatggg ccgacatacg tgggaaagca ttggacgccc actgccagga aggaagaata
     3361 taatcctgtc atcacagccg tccacggacg atagagtcac ttgggtaaaa tccgttgacg
     3421 aagccatagc tgcctgtggt gatgtgcctg agataatggt aataggagga gggcgggtaa
     3481 tcgaacaatt tctgcctaaa gcccagaagc tgtaccttac ccatattgac gcggaagtag
     3541 agggcgacac acatttccca gattatgagc cagatgattg ggagagcgtt ttctccgaat
     3601 ttcatgatgc agacgcccag aatagccaca gctactgctt tgaaatattg gagcgccgat
     3661 gaagctagcg aattcgaatt taaatcggcc tcgactgtgc cttctagttg ccagccatct
     3721 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3781 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3841 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3901 gacgcggccg caatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3961 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4021 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4081 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4141 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4201 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4261 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
     4321 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
     4381 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
     4441 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
     4501 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
     4561 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
     4621 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
     4681 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
     4741 ccgaggagca ggactgagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     4801 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     4861 tgtatcatgc gttaactaaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4921 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4981 aaactcatca atgtatctta tcatgtctgg aattgactca aatgatgtca attagtctat
     5041 cagaagctca tctggtctcc cttccggggg acaagacatc cctgtttaat atttaaacag
     5101 cagtgttccc aaactgggtt cttatatccc ttgctctggt caaccaggtt gcagggtttc
     5161 ctgtcctcac aggaacgaag tccctaaaga aacagtggca gccaggttta gccccggaat
     5221 tgactggatt ccttttttag ggcccattgg tatggctttt tccccgtatc cccccaggtg
     5281 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
     5341 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
     5401 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
     5461 ctgggggctt tggggggggg ctgtccctga tatctataac aagaaaatat atatataata
     5521 agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa
     5581 aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat
     5641 cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat
     5701 gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa
     5761 tgcatgcgtc aattttacgc agactatctt tctagggtta atctagctgc atcaggatca
     5821 tatcgtcggg tcttttttcc ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg
     5881 ggaggaagaa gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag agtttgccga
     5941 ggatgactgc tgctgcattg acgttgagcg aaaacgcacg tttaccatga tgattcggga
     6001 aggtgtggcc atgcacgcct ttaacggtga actgttcgtt caggccacct gggataccag
     6061 ttcgtcgcgg cttttccgga cacagttccg gatggtcagc ccgaagcgca tcagcaaccc
     6121 gaacaatacc ggcgacagcc ggaactgccg tgccggtgtg cagattaatg acagcggtgc
     6181 ggcgctggga tattacgtca gcgaggacgg gtatcctggc tggatgccgc agaaatggac
     6241 atggataccc cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc atagctgttt
     6301 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     6361 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     6421 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6481 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6541 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6601 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6661 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6721 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6781 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6841 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6901 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6961 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7021 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7081 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     7141 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7201 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7261 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7321 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7381 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7441 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7501 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7561 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7621 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7681 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7741 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7801 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7861 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7921 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7981 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     8041 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     8101 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     8161 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     8221 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     8281 agggcgacac ggaaatgttg aatactcat
//
