LOCUS       sequence-224818-        7777 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 7777)
  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 7777)
  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-TO-Casp3te-tv-cx
FEATURES             Location/Qualifiers
     source          1..7777
                     /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"
     protein_bind    1994..2012
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2015..2033
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2043..2067
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2159..2168
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2693..2713
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3032..3052
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     primer_bind     complement(3157..3174)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3163..3370
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3403..3423)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3408..3737
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3588..3723
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3650..3669
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3785..3832
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3851..4225
                     /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(4285..4305)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4347..4468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4384..4403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4438..4457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5231..5265
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5756..5772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5756..5772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5769..5791)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5780..5796
                     /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(5804..5810,5811..5828,5829..5834))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5849..5870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5987..6004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6158..6746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6238..6257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6917..7708,7709..7777))
                     /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     7540..7559
                     /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 ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcga
     2041 cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
     2101 catagaagac accgggaccg atccagcctc cggactctag cgtttaaact taagcttcgc
     2161 caccatggga tccgaaaaca ctgagaacag cgtcgatagc aagtccatta agaacctcga
     2221 acctaaaatc atacatggca gtgagagcat ggatagcggc ataagccttg acaactccta
     2281 taaaatggac tacccggaga tgggtctgtg cataataatc aacaacaaga acttccacaa
     2341 atccacgggg atgacaagta ggtctggaac cgatgtcgat gcggcaaact tgcgcgagac
     2401 ctttagaaac ctcaagtacg aggtacgcaa taaaaacgat ctcactcgcg aggaaatagt
     2461 agagctgatg cgagatgtca gtaaggaaga tcattccaag agatcctcct tcgtctgcgt
     2521 cttgttgagt cacggagagg aagggatcat attcggtacg aacggcccag tggatttgaa
     2581 aaagataacc aatttcttta gaggtgaccg gtgccgctct ctcacaggaa agccaaaact
     2641 ctttattatc caagcatgta ggggtacgga actggattgt ggaatcgaaa ctgagaactt
     2701 gtacttccag tccggggtag atgacgacat ggcctgccat aaaatccctg tcgaggccga
     2761 ttttctctac gcctactcta ctgcgcctgg gtattacagc tggagaaaca gcaaggatgg
     2821 ttcctggttt atacaaagcc tgtgtgctat gctcaaacag tatgcggata agctggagtt
     2881 catgcatatc cttaccagag taaatcgcaa agtggccaca gagtttgagt cattttcttt
     2941 tgacgcaaca tttcacgcca agaagcagat tccttgtata gtatctatgc tgacgaaaga
     3001 gctgtacttc tatcacagca ccggcggatc tgaaaccgtg cggtttcaat ccagcggcgg
     3061 tggaggacgg agcaaactga atccacccga tgagtctgga cccggttgta tgtcatgtaa
     3121 atgtgtgttg agctagcgaa ttcgaattta aatcggcctc gactgtgcct tctagttgcc
     3181 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3241 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3301 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3361 atgctgggga cgcggccgca atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3421 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3481 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3541 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3601 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3661 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3721 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     3781 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3841 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     3901 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     3961 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     4021 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     4081 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     4141 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     4201 cttcgtggcc gaggagcagg actgagtcga caatcaacct ctggattaca aaatttgtga
     4261 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4321 aatgcctttg tatcatgcgt taactaaact tgtttattgc agcttataat ggttacaaat
     4381 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4441 gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat
     4501 tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat
     4561 ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc
     4621 agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc
     4681 cccggaattg actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc
     4741 cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc
     4801 caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg
     4861 ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa
     4921 ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat
     4981 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     5041 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     5101 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     5161 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     5221 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat
     5281 caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg
     5341 ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag
     5401 tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg
     5461 attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg
     5521 gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc
     5581 agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac
     5641 agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag
     5701 aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat
     5761 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5821 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5881 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5941 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6001 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6061 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6121 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6181 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6241 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6301 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6361 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6421 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6481 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6541 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6601 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6661 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6721 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6781 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6841 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6901 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6961 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7021 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7081 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7141 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7201 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7261 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7321 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7381 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7441 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7501 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7561 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7621 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7681 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7741 agggaataag ggcgacacgg aaatgttgaa tactcat
//
