LOCUS       sequence-225021-        7398 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 7398)
  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 7398)
  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-tvD-Cit
FEATURES             Location/Qualifiers
     source          1..7398
                     /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             join(2031..2033,2034..2750)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYKS"
     primer_bind     complement(2073..2094)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2334..2353)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2681..2702
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2778..2795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2784..2991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3024..3044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3029..3358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3209..3344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3271..3290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3406..3453
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3472..3846
                     /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(3906..3926)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3968..4089
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4005..4024)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4059..4078
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4852..4886
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5377..5393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5377..5393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5390..5412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5401..5417
                     /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(5425..5431,5432..5449,5450..5455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5470..5491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5608..5625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5779..6367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5859..5878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6538..7329,7330..7398))
                     /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     7161..7180
                     /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 ggatccggag gtagtgagac ggttagattc cagtaccaca
     1981 aatctggagc ctggaaactt ccggtgagtc tggtaaaggg cggtacctca gtgagcaagg
     2041 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2101 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2161 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     2221 tcggctacgg cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     2281 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2341 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2401 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2461 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     2521 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     2581 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc
     2641 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2701 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagagctag cagctagcga
     2761 attcgaattt aaatcggcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2821 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2881 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2941 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg acgcggccgc
     3001 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3121 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3181 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3241 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3301 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3361 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     3421 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     3481 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     3541 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     3601 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3661 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3721 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3781 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3841 gactgagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3901 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgcg
     3961 ttaactaaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4021 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4081 tgtatcttat catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat
     4141 ctggtctccc ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca
     4201 aactgggttc ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca
     4261 ggaacgaagt ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc
     4321 cttttttagg gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc
     4381 aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg
     4441 ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc
     4501 cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt
     4561 gggggggggc tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt
     4621 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     4681 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     4741 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     4801 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     4861 attttacgca gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt
     4921 cttttttccg gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag
     4981 cccgtgcctt ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct
     5041 gctgcattga cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca
     5101 tgcacgcctt taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc
     5161 ttttccggac acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg
     5221 gcgacagccg gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat
     5281 attacgtcag cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc
     5341 gtgagttacc cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5401 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5461 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5521 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5581 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7381 gaaatgttga atactcat
//
