LOCUS       sequence-225370-        7832 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 7832)
  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 7832)
  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-Cit-tevs-DHFR
FEATURES             Location/Qualifiers
     source          1..7832
                     /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(1947..1949,1950..2660)
                     /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(1989..2010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2250..2269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2597..2618
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2676..2696
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3212..3229)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3218..3425
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3458..3478)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3463..3792
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3643..3778
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3705..3724
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3840..3887
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3906..4280
                     /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(4340..4360)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4402..4523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4439..4458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4493..4512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5286..5320
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5811..5827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5811..5827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5824..5846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5835..5851
                     /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(5859..5865,5866..5883,5884..5889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5904..5925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6042..6059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6213..6801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6293..6312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6972..7763,7764..7832))
                     /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     7595..7614
                     /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 ggatccgtga gcaagggcga ggagctgttc accggggtgg
     1981 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2041 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2101 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg atgtgcttcg
     2161 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2221 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2281 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2341 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2401 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2461 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2521 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca
     2581 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2641 gcatggacga gctgtacaag ggtgggtccg gtctcgagaa cttgtacttc cagagtggga
     2701 gcggatcgat atcactgatc gctgcactcg cggttgatta tgtaataggc atggaaaacg
     2761 cgatgccctg gaacctgcca gcagacctcg catggtttaa gcggaacact ctgaataagc
     2821 cagtcattat gggccgacat acgtgggaaa gcattggacg cccactgcca ggaaggaaga
     2881 atataatcct gtcatcacag ccgtccacgg acgatagagt cacttgggta aaatccgttg
     2941 acgaagccat agctgcctgt ggtgatgtgc ctgagataat ggtaatagga ggagggcggg
     3001 taatcgaaca atttctgcct aaagcccaga agctgtacct tacccatatt gacgcggaag
     3061 tagagggcga cacacatttc ccagattatg agccagatga ttgggagagc gttttctccg
     3121 aatttcatga tgcagacgcc cagaatagcc acagctactg ctttgaaata ttggagcgcc
     3181 gatgaagcta gcgaattcga atttaaatcg gcctcgactg tgccttctag ttgccagcca
     3241 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3301 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3361 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3421 ggggacgcgg ccgcaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3541 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3601 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3661 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3721 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3781 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     3841 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     3901 aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     3961 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     4021 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     4081 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     4141 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     4201 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     4261 tggccgagga gcaggactga gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     4321 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     4381 ctttgtatca tgcgttaact aaacttgttt attgcagctt ataatggtta caaataaagc
     4441 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4501 tccaaactca tcaatgtatc ttatcatgtc tggaattgac tcaaatgatg tcaattagtc
     4561 tatcagaagc tcatctggtc tcccttccgg gggacaagac atccctgttt aatatttaaa
     4621 cagcagtgtt cccaaactgg gttcttatat cccttgctct ggtcaaccag gttgcagggt
     4681 ttcctgtcct cacaggaacg aagtccctaa agaaacagtg gcagccaggt ttagccccgg
     4741 aattgactgg attccttttt tagggcccat tggtatggct ttttccccgt atccccccag
     4801 gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct
     4861 tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga
     4921 ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca
     4981 tccctggggg ctttgggggg gggctgtccc tgatatctat aacaagaaaa tatatatata
     5041 ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct
     5101 taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa
     5161 aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga
     5221 gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa
     5281 gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga
     5341 tcatatcgtc gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat
     5401 cggggaggaa gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc
     5461 cgaggatgac tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg
     5521 ggaaggtgtg gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac
     5581 cagttcgtcg cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa
     5641 cccgaacaat accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg
     5701 tgcggcgctg ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg
     5761 gacatggata ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg
     5821 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5881 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5941 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6001 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6061 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6121 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6181 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6241 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6301 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6361 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6421 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6481 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6541 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6601 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6661 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6721 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6781 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6841 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6901 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6961 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7021 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7081 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7141 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7201 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7261 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7321 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7381 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7441 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7501 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7561 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7621 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7681 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7741 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7801 ataagggcga cacggaaatg ttgaatactc at
//
