LOCUS       sequence-225388-        8012 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 8012)
  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 8012)
  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-ZhcD-Cit-tevs-DHFR
FEATURES             Location/Qualifiers
     source          1..8012
                     /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(2127..2129,2130..2840)
                     /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(2169..2190)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2430..2449)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2777..2798
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2856..2876
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3392..3409)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3398..3605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3638..3658)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3643..3972
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3823..3958
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3885..3904
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4020..4067
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4086..4460
                     /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(4520..4540)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4582..4703
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4619..4638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4673..4692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5466..5500
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5991..6007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5991..6007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6004..6026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6015..6031
                     /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(6039..6045,6046..6063,6064..6069))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6084..6105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6222..6239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6393..6981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6473..6492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7152..7943,7944..8012))
                     /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     7775..7794
                     /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 ggatccgagc aactcgaaaa gaagctgcaa gcgttggaga
     1981 agaagctcgc acaacttgaa tggaaaaatc aggcactgga aaagaaactg gctcaaggtg
     2041 gcggaggtag tgaggatgtt gtttgttgcc accacaaatc tggagcctgg aaacttccgg
     2101 tgagtctggt aaagggtggg ggttcagtga gcaagggcga ggagctgttc accggggtgg
     2161 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2221 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2281 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg atgtgcttcg
     2341 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2401 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2461 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2521 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2581 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2641 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2701 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca
     2761 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2821 gcatggacga gctgtacaag ggtgggtccg gtctcgagaa cttgtacttc cagagtggga
     2881 gcggatcgat atcactgatc gctgcactcg cggttgatta tgtaataggc atggaaaacg
     2941 cgatgccctg gaacctgcca gcagacctcg catggtttaa gcggaacact ctgaataagc
     3001 cagtcattat gggccgacat acgtgggaaa gcattggacg cccactgcca ggaaggaaga
     3061 atataatcct gtcatcacag ccgtccacgg acgatagagt cacttgggta aaatccgttg
     3121 acgaagccat agctgcctgt ggtgatgtgc ctgagataat ggtaatagga ggagggcggg
     3181 taatcgaaca atttctgcct aaagcccaga agctgtacct tacccatatt gacgcggaag
     3241 tagagggcga cacacatttc ccagattatg agccagatga ttgggagagc gttttctccg
     3301 aatttcatga tgcagacgcc cagaatagcc acagctactg ctttgaaata ttggagcgcc
     3361 gatgaagcta gcgaattcga atttaaatcg gcctcgactg tgccttctag ttgccagcca
     3421 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3481 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3541 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3601 ggggacgcgg ccgcaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3661 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3721 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3781 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3841 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3901 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3961 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     4021 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     4081 aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     4141 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     4201 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     4261 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     4321 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     4381 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     4441 tggccgagga gcaggactga gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     4501 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     4561 ctttgtatca tgcgttaact aaacttgttt attgcagctt ataatggtta caaataaagc
     4621 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4681 tccaaactca tcaatgtatc ttatcatgtc tggaattgac tcaaatgatg tcaattagtc
     4741 tatcagaagc tcatctggtc tcccttccgg gggacaagac atccctgttt aatatttaaa
     4801 cagcagtgtt cccaaactgg gttcttatat cccttgctct ggtcaaccag gttgcagggt
     4861 ttcctgtcct cacaggaacg aagtccctaa agaaacagtg gcagccaggt ttagccccgg
     4921 aattgactgg attccttttt tagggcccat tggtatggct ttttccccgt atccccccag
     4981 gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct
     5041 tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga
     5101 ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca
     5161 tccctggggg ctttgggggg gggctgtccc tgatatctat aacaagaaaa tatatatata
     5221 ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct
     5281 taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa
     5341 aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga
     5401 gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa
     5461 gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga
     5521 tcatatcgtc gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat
     5581 cggggaggaa gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc
     5641 cgaggatgac tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg
     5701 ggaaggtgtg gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac
     5761 cagttcgtcg cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa
     5821 cccgaacaat accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg
     5881 tgcggcgctg ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg
     5941 gacatggata ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg
     6001 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6061 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6121 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6181 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6241 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6301 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6361 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6421 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6481 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6541 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6601 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6661 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6721 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6781 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6841 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6901 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6961 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     7021 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     7081 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7141 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7201 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7261 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7321 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7381 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7441 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7501 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7561 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7621 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7681 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7741 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7801 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7861 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7921 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7981 ataagggcga cacggaaatg ttgaatactc at
//
