LOCUS       sequence-152155-        5886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Toggle switch plasmid containing lacI-pdt3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5886)
  AUTHORS   Cameron DE, Collins JJ
  TITLE     Tunable protein degradation in bacteria.
  JOURNAL   Nat Biotechnol. 2014 Dec;32(12):1276-81. doi: 10.1038/nbt.3053. Epub
            2014 Nov 17.
  PUBMED    25402616
REFERENCE   2  (bases 1 to 5886)
  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: "10.1038/nbt.3053"; 
            journalName: "Nat Biotechnol"; date: "2014-12"; volume: "32"; issue:
            "12"; pages: "1276-81"
COMMENT     Sequence Label: pECJ3
FEATURES             Location/Qualifiers
     source          1..5886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(58..771)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(98..118)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     713..741
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(join(823..848,849..854,855..871,872..877,
                     878..896))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    853..871
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    878..896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(1023..2102)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2064..2083
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(2148..2173,2174..2179,2180..2196,2197..2202,
                     2203..2221))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2178..2196
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2203..2221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        join(2253..2258,2259..2275,2276..2282)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2290..2306
                     /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)."
     CDS             2353..2976
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2426..2445)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(3031..3036,3037..3053,3054..3060)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3068..3084
                     /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)."
     CDS             3135..3845
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3280..3298)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3548..3567
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3905..3991
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4155..4743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4235..4254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4831..4925)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4956..5750)
                     /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(5067..5086)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5677..5696
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtctgtg caagtactac tgttctgcag tcacttgaat tcgataccca gctgttattt
       61 gtatagttca tccatgccat gtgtaatccc agcagcggtt acaaactcaa gaaggaccat
      121 gtggtctctc ttttcgttgg gatctttcga aagggcagat tgtgtggaca ggtaatggtt
      181 gtctggtaaa aggacagggc catcgccaat cggagtattt tgttgataat ggtctgctag
      241 ttgaacgctt ccatcttcaa tgttgtgtct aattttgaag ttaactttga ttccattctt
      301 ttgtttgtct gccatgatgt atacattgtg tgagttatag ttgtattcca atttgtgtcc
      361 aagaatgttt ccatcttctt taaaatcaat accttttaac tcgattctat taacaagggt
      421 atcaccttca aacttgactt cagcacgtgt cttgtagttc ccgtcatctt tgaaaaatat
      481 agttctttcc tgtacgtaac cttcgggcat ggcactcttg aaaaagtcat gctgtttcat
      541 gtgatctggg tatctcgcaa agcattgaac accataaccg aaagtagtga caagtgttgg
      601 ccacggaaca ggtagttttc cagtagtgca aataaattta agggtaagtt ttccgtatgt
      661 tgcatcacct tcaccctctc cactgacaga aaatttgtgc ccattaacat caccatctaa
      721 ttcaacaaga attgggacaa ctccagtgaa aagttcttct cctttacgca tgtgcactac
      781 ctccttggtt gtctatgcta tgctgatcta caactggcat gcggtcagtg cgtcctgctg
      841 atgtgctcag tatctctatc actgataggg atgtcaatct ctatcactga tagggaaacg
      901 tttcgcagaa gcttccgcaa ggtaccactt tgccgcggtt aaactcgtct tctgttcgcc
      961 tggcccgcgt tcagcataaa ggtcggcact tcgttggtgt tttcttcgtt tttgttcgcc
     1021 gcctgcccgc tttccagtcg ggaaacctgt cgtgccagtt gcattaatga atcggccaac
     1081 gcgcggggag aggcggtttg cgtattggga gccagggtgg tttttctttt caccagtgag
     1141 acgggcaaca gttgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     1201 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     1261 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     1321 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     1381 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     1441 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     1501 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     1561 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     1621 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     1681 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     1741 ccactgacgc gctgcgcgag aagattgtga accgctgctt tacaggcttc gacgccgctt
     1801 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     1861 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     1921 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     1981 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     2041 gaaacggtct gataagagac accggcatac tctgcgacat cgtacagcgt tactggtttc
     2101 acattcacca ttgtacaccc tctccttata ttgggaccta tggatccggt cagtgcgtcc
     2161 tgctgatgtg ctcagtatct ctatcactga tagggatgtc aatctctatc actgataggg
     2221 acggccgccc gttccatggc tgaaatgagc tgttgacaat taatcatccg gctcgtataa
     2281 tgtgtggaat tgtgagcgga taacaatttc acacgtcgac cgccttcggc gaagctccac
     2341 acgagagcta gcatgtctcg tttagataaa agtaaagtga ttaacagcgc attagagctg
     2401 cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa gctaggtgta
     2461 gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga cgccttagcc
     2521 attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga aagctggcaa
     2581 gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca tcgcgatgga
     2641 gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct cgaaaatcaa
     2701 ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc actcagcgct
     2761 gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa
     2821 gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc tatcgaatta
     2881 tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat catctgcgga
     2941 ttagaaaaac aacttaaatg tgaaagtggg tcttgagagc tcggactgct taagtcgctc
     3001 catatgctcg ttcccgggcg aaatgagctg ttgacaatta atcatccggc tcgtataatg
     3061 tgtggaattg tgagcggata acaatttcac accaattgag tattaactat cgttcaactg
     3121 atagggaggc gccgatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     3181 tcatgcgctt caaggttcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     3241 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     3301 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     3361 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     3421 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     3481 cctccctgca agacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     3541 ccgacggccc cgtaatgcag aagaagacta tgggctggga ggcctcctcc gagcggatgt
     3601 accccgagga cggcgcgctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     3661 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg caactgcccg
     3721 gcgcgtacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     3781 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtata
     3841 agtaagatat ctatcgccct agggaccgtc tcgagagaat caatattaat ccaacgcgtg
     3901 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     3961 gtcggtgaac gctctcctga gtaggacaaa tccgccgccc tagacttagg cgttcggctg
     4021 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4081 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4141 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4201 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4261 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4321 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4381 taggtcgttc gctccaagct gggctgtctg cacgaacccc ccgttcagcc cgaccgctgc
     4441 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4501 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4561 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4621 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4681 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4741 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4801 taagggattt tggtcatggc tagtgcttgg attctcacca ataaaaaacg cccggcggca
     4861 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     4921 gagtccaagc caattctcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     4981 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     5041 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     5101 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     5161 atgatattcg gcaagcaggc atcgccgtgg gtcacgacga gatcctcgcc gtcgggcata
     5221 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     5281 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     5341 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     5401 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     5461 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagccgcg
     5521 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc
     5581 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gaccctgcgc tgacagccgg
     5641 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     5701 tccacccaag ccgccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     5761 cctcatcctg tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag
     5821 aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgg cccaactggc
     5881 aattcc
//
