LOCUS       sequence-225375-        8235 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 8235)
  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 8235)
  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-tehc-DHFR
FEATURES             Location/Qualifiers
     source          1..8235
                     /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(3242..3259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3248..3455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3488..3508)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3493..3822
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3673..3808
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3735..3754
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3889..4683
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3943..3962)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4553..4572
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4743..4763)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4805..4926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4842..4861)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4896..4915
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5689..5723
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6214..6230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6214..6230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6227..6249)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6238..6254
                     /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(6262..6268,6269..6286,6287..6292))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6307..6328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6445..6462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6616..7204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6696..6715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7375..8166,8167..8235))
                     /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     7998..8017
                     /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 cagagtgacg
     2701 agatggagga gtgtagtcag caccttggga gcggatcgat atcactgatc gctgcactcg
     2761 cggttgatta tgtaataggc atggaaaacg cgatgccctg gaacctgcca gcagacctcg
     2821 catggtttaa gcggaacact ctgaataagc cagtcattat gggccgacat acgtgggaaa
     2881 gcattggacg cccactgcca ggaaggaaga atataatcct gtcatcacag ccgtccacgg
     2941 acgatagagt cacttgggta aaatccgttg acgaagccat agctgcctgt ggtgatgtgc
     3001 ctgagataat ggtaatagga ggagggcggg taatcgaaca atttctgcct aaagcccaga
     3061 agctgtacct tacccatatt gacgcggaag tagagggcga cacacatttc ccagattatg
     3121 agccagatga ttgggagagc gttttctccg aatttcatga tgcagacgcc cagaatagcc
     3181 acagctactg ctttgaaata ttggagcgcc gatgaagcta gcgaattcga atttaaatcg
     3241 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3301 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3361 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3421 gaggattggg aagacaatag caggcatgct ggggacgcgg ccgcaattta acgcgaatta
     3481 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3541 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3601 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3661 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3721 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     3781 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     3841 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3901 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3961 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4021 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4081 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4141 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4201 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4261 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4321 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4381 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4441 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4501 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4561 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4621 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4681 tgagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4741 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgcgtta
     4801 actaaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4861 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4921 atcttatcat gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg
     4981 gtctcccttc cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac
     5041 tgggttctta tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga
     5101 acgaagtccc taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt
     5161 ttttagggcc cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa
     5221 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg
     5281 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg
     5341 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg
     5401 ggggggctgt ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag
     5461 tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag
     5521 ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc
     5581 gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca
     5641 gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt
     5701 ttacgcagac tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt
     5761 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc
     5821 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct
     5881 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc
     5941 acgcctttaa cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt
     6001 tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg
     6061 acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt
     6121 acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg
     6181 agttacccgg cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     6241 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6301 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6361 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6421 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6481 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6541 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6601 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6661 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6721 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6781 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6841 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6901 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6961 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7021 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7081 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7141 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7201 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7261 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7321 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7381 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7441 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7501 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7561 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7621 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7681 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7741 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7801 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7861 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7921 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7981 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8041 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8101 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8161 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8221 atgttgaata ctcat
//
