LOCUS       sequence-225010-        7992 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 7992)
  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 7992)
  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-CMV-Cit-tvmvs-DHFR
FEATURES             Location/Qualifiers
     source          1..7992
                     /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)"
     enhancer        1409..1788
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1789..1992
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1943..1963
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2037..2056
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2037..2055
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2092..2101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2107..2109,2110..2820)
                     /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(2149..2170)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2410..2429)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2757..2778
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2836..2856
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     primer_bind     complement(3372..3389)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3378..3585
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3618..3638)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3623..3952
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3803..3938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3865..3884
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4000..4047
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4066..4440
                     /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(4500..4520)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4562..4683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4599..4618)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4653..4672
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5446..5480
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5971..5987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5971..5987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5984..6006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5995..6011
                     /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(6019..6025,6026..6043,6044..6049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6064..6085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6202..6219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6373..6961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6453..6472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7132..7923,7924..7992))
                     /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     7755..7774
                     /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 tacgcgttga cattgattat tgactagtta ttaatagtaa
     1441 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1501 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1561 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1621 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     1681 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1741 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1801 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1861 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1921 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1981 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     2041 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt cgccaccatg
     2101 ggatccgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2161 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2221 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2281 accctcgtga ccaccttcgg ctacggcctg atgtgcttcg cccgctaccc cgaccacatg
     2341 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2401 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2461 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2521 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2581 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2641 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2701 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2761 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2821 ggtgggtccg gtctcgagac ggttagattc cagagtggga gcggatcgat atcactgatc
     2881 gctgcactcg cggttgatta tgtaataggc atggaaaacg cgatgccctg gaacctgcca
     2941 gcagacctcg catggtttaa gcggaacact ctgaataagc cagtcattat gggccgacat
     3001 acgtgggaaa gcattggacg cccactgcca ggaaggaaga atataatcct gtcatcacag
     3061 ccgtccacgg acgatagagt cacttgggta aaatccgttg acgaagccat agctgcctgt
     3121 ggtgatgtgc ctgagataat ggtaatagga ggagggcggg taatcgaaca atttctgcct
     3181 aaagcccaga agctgtacct tacccatatt gacgcggaag tagagggcga cacacatttc
     3241 ccagattatg agccagatga ttgggagagc gttttctccg aatttcatga tgcagacgcc
     3301 cagaatagcc acagctactg ctttgaaata ttggagcgcc gatgaagcta gcgaattcga
     3361 atttaaatcg gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3421 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3481 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3541 cagcaagggg gaggattggg aagacaatag caggcatgct ggggacgcgg ccgcaattta
     3601 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3661 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3721 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3781 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3841 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3901 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3961 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     4021 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc
     4081 agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac
     4141 cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac
     4201 gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg
     4261 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac
     4321 ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag
     4381 ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga
     4441 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4501 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgcgttaact
     4561 aaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4621 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4681 ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc
     4741 tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg
     4801 gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg
     4861 aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt
     4921 tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag
     4981 cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc
     5041 ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg
     5101 gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg
     5161 gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
     5221 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
     5281 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
     5341 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
     5401 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
     5461 cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt
     5521 tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg
     5581 ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca
     5641 ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg
     5701 cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc
     5761 ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca
     5821 gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg
     5881 tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt
     5941 tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6001 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6061 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6121 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6181 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6241 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6301 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6361 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6421 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6481 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6541 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6601 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6661 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6721 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6781 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6841 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6901 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6961 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7021 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7081 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7141 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7201 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7261 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7321 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7381 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7441 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7501 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7561 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7621 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7681 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7741 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7801 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7861 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7921 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7981 ttgaatactc at
//
