LOCUS       sequence-225019-        8214 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 8214)
  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 8214)
  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-hcvs-DHFR
FEATURES             Location/Qualifiers
     source          1..8214
                     /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"
     primer_bind     complement(3221..3238)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3227..3434
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3467..3487)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3472..3801
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3652..3787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3714..3733
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3868..4662
                     /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(3922..3941)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4532..4551
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4722..4742)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4784..4905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4821..4840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4875..4894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5668..5702
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6193..6209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6193..6209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6206..6228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6217..6233
                     /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(6241..6247,6248..6265,6266..6271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6286..6307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6424..6441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6595..7183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6675..6694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7354..8145,8146..8214))
                     /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     7977..7996
                     /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 gtctcgacga gatggaggag tgtagtcagc
     2701 accttgggag cggatcgata tcactgatcg ctgcactcgc ggttgattat gtaataggca
     2761 tggaaaacgc gatgccctgg aacctgccag cagacctcgc atggtttaag cggaacactc
     2821 tgaataagcc agtcattatg ggccgacata cgtgggaaag cattggacgc ccactgccag
     2881 gaaggaagaa tataatcctg tcatcacagc cgtccacgga cgatagagtc acttgggtaa
     2941 aatccgttga cgaagccata gctgcctgtg gtgatgtgcc tgagataatg gtaataggag
     3001 gagggcgggt aatcgaacaa tttctgccta aagcccagaa gctgtacctt acccatattg
     3061 acgcggaagt agagggcgac acacatttcc cagattatga gccagatgat tgggagagcg
     3121 ttttctccga atttcatgat gcagacgccc agaatagcca cagctactgc tttgaaatat
     3181 tggagcgccg atgaagctag cgaattcgaa tttaaatcgg cctcgactgt gccttctagt
     3241 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3301 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3361 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3421 aggcatgctg gggacgcggc cgcaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3481 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3541 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3601 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3661 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3721 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3781 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3841 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3901 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3961 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4021 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4081 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4141 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4201 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4261 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4321 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4381 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4441 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4501 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4561 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4621 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagtcgacaa tcaacctctg
     4681 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     4741 tgtggatacg ctgctttaat gcctttgtat catgcgttaa ctaaacttgt ttattgcagc
     4801 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4861 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggaattg
     4921 actcaaatga tgtcaattag tctatcagaa gctcatctgg tctcccttcc gggggacaag
     4981 acatccctgt ttaatattta aacagcagtg ttcccaaact gggttcttat atcccttgct
     5041 ctggtcaacc aggttgcagg gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag
     5101 tggcagccag gtttagcccc ggaattgact ggattccttt tttagggccc attggtatgg
     5161 ctttttcccc gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga
     5221 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg
     5281 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta
     5341 gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cctgatatct
     5401 ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat
     5461 aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac
     5521 tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac
     5581 gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta
     5641 gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag
     5701 ggttaatcta gctgcatcag gatcatatcg tcgggtcttt tttccggctc agtcatcgcc
     5761 caagctggcg ctatctgggc atcggggagg aagaagcccg tgccttttcc cgcgaggttg
     5821 aagcggcatg gaaagagttt gccgaggatg actgctgctg cattgacgtt gagcgaaaac
     5881 gcacgtttac catgatgatt cgggaaggtg tggccatgca cgcctttaac ggtgaactgt
     5941 tcgttcaggc cacctgggat accagttcgt cgcggctttt ccggacacag ttccggatgg
     6001 tcagcccgaa gcgcatcagc aacccgaaca ataccggcga cagccggaac tgccgtgccg
     6061 gtgtgcagat taatgacagc ggtgcggcgc tgggatatta cgtcagcgag gacgggtatc
     6121 ctggctggat gccgcagaaa tggacatgga taccccgtga gttacccggc gggcgcgctt
     6181 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     6241 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     6301 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6361 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7021 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcat
//
