LOCUS       sequence-225017-        7494 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 7494)
  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 7494)
  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
FEATURES             Location/Qualifiers
     source          1..7494
                     /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..2846)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYKS"
     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"
     primer_bind     complement(2874..2891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2880..3087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3120..3140)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3125..3454
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3305..3440
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3367..3386
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3502..3549
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3568..3942
                     /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(4002..4022)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4064..4185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4101..4120)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4155..4174
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4948..4982
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5473..5489)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5473..5489)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5486..5508)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5497..5513
                     /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(5521..5527,5528..5545,5546..5551))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5566..5587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5704..5721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5875..6463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5955..5974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6634..7425,7426..7494))
                     /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     7257..7276
                     /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 agctagcagc tagcgaattc gaatttaaat cggcctcgac
     2881 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2941 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3001 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3061 ggaagacaat agcaggcatg ctggggacgc ggccgcaatt taacgcgaat taattctgtg
     3121 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3181 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3241 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3301 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3361 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3421 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3481 tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     3541 taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc
     3601 accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg
     3661 gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
     3721 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
     3781 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
     3841 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
     3901 gccggcaact gcgtgcactt cgtggccgag gagcaggact gagtcgacaa tcaacctctg
     3961 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     4021 tgtggatacg ctgctttaat gcctttgtat catgcgttaa ctaaacttgt ttattgcagc
     4081 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4141 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggaattg
     4201 actcaaatga tgtcaattag tctatcagaa gctcatctgg tctcccttcc gggggacaag
     4261 acatccctgt ttaatattta aacagcagtg ttcccaaact gggttcttat atcccttgct
     4321 ctggtcaacc aggttgcagg gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag
     4381 tggcagccag gtttagcccc ggaattgact ggattccttt tttagggccc attggtatgg
     4441 ctttttcccc gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga
     4501 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg
     4561 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta
     4621 gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cctgatatct
     4681 ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat
     4741 aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac
     4801 tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac
     4861 gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta
     4921 gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag
     4981 ggttaatcta gctgcatcag gatcatatcg tcgggtcttt tttccggctc agtcatcgcc
     5041 caagctggcg ctatctgggc atcggggagg aagaagcccg tgccttttcc cgcgaggttg
     5101 aagcggcatg gaaagagttt gccgaggatg actgctgctg cattgacgtt gagcgaaaac
     5161 gcacgtttac catgatgatt cgggaaggtg tggccatgca cgcctttaac ggtgaactgt
     5221 tcgttcaggc cacctgggat accagttcgt cgcggctttt ccggacacag ttccggatgg
     5281 tcagcccgaa gcgcatcagc aacccgaaca ataccggcga cagccggaac tgccgtgccg
     5341 gtgtgcagat taatgacagc ggtgcggcgc tgggatatta cgtcagcgag gacgggtatc
     5401 ctggctggat gccgcagaaa tggacatgga taccccgtga gttacccggc gggcgcgctt
     5461 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5521 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5581 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5641 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5701 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5761 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5821 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5881 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5941 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6001 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6061 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6121 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6181 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6241 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6301 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6361 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6421 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6481 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6541 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6601 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6661 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6721 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6781 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6841 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6901 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6961 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7021 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7081 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7141 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7201 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7261 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7321 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7381 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7441 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcat
//
