LOCUS       sequence-225387-        7925 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 7925)
  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 7925)
  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-teD-Cit-hcvs-DHFR
FEATURES             Location/Qualifiers
     source          1..7925
                     /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..2744)
                     /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(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(3305..3322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3311..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3551..3571)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3556..3885
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3736..3871
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3798..3817
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3933..3980
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3999..4373
                     /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(4433..4453)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4495..4616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4532..4551)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4586..4605
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5379..5413
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5904..5920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5904..5920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5917..5939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5928..5944
                     /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(5952..5958,5959..5976,5977..5982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5997..6018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6135..6152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6306..6894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6386..6405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7065..7856,7857..7925))
                     /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     7688..7707
                     /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 gtagtgagaa cctttatttc cagtaccaca
     1981 aatctggagc ctggaaactt ccggtgagtc tggtaaaggg tgggggttca 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 caagggtggg tccggtctcg
     2761 acgagatgga ggagtgtagt cagcaccttg ggagcggatc gatatcactg atcgctgcac
     2821 tcgcggttga ttatgtaata ggcatggaaa acgcgatgcc ctggaacctg ccagcagacc
     2881 tcgcatggtt taagcggaac actctgaata agccagtcat tatgggccga catacgtggg
     2941 aaagcattgg acgcccactg ccaggaagga agaatataat cctgtcatca cagccgtcca
     3001 cggacgatag agtcacttgg gtaaaatccg ttgacgaagc catagctgcc tgtggtgatg
     3061 tgcctgagat aatggtaata ggaggagggc gggtaatcga acaatttctg cctaaagccc
     3121 agaagctgta ccttacccat attgacgcgg aagtagaggg cgacacacat ttcccagatt
     3181 atgagccaga tgattgggag agcgttttct ccgaatttca tgatgcagac gcccagaata
     3241 gccacagcta ctgctttgaa atattggagc gccgatgaag ctagcgaatt cgaatttaaa
     3301 tcggcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3361 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     3421 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3481 ggggaggatt gggaagacaa tagcaggcat gctggggacg cggccgcaat ttaacgcgaa
     3541 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3661 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3721 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3781 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3841 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3901 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
     3961 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
     4021 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     4081 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     4141 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     4201 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     4261 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     4321 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgagtcgaca
     4381 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4441 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgcgtta actaaacttg
     4501 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4561 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4621 gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg gtctcccttc
     4681 cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac tgggttctta
     4741 tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga acgaagtccc
     4801 taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt ttttagggcc
     4861 cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     4921 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     4981 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     5041 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     5101 ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
     5161 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
     5221 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
     5281 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
     5341 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
     5401 tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct
     5461 cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc
     5521 ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt
     5581 tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa
     5641 cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca
     5701 gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa
     5761 ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga
     5821 ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg
     5881 cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5941 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     6001 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6061 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6121 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6301 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6901 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6961 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7021 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7081 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7141 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7201 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7261 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7321 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7381 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7441 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7501 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7561 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7621 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7681 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7741 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7801 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7861 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7921 ctcat
//
