LOCUS       sequence-68819-e        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rtTA positive feedback loop with three tet operators in the GAL1 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  AUTHORS   Hsu C, Scherrer S, Buetti-Dinh A, Ratna P, Pizzolato J, Jaquet V, 
            Becskei A
  TITLE     Stochastic signalling rewires the interaction map of a multiple 
            feedback network during yeast evolution.
  JOURNAL   Nat Commun. 2012 Feb 21;3:682. doi: 10.1038/ncomms1687.
  PUBMED    22353713
REFERENCE   2  (bases 1 to 6958)
  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"; journalName: "Nat 
            Commun."; date: "2012-02-21"; volume: "3:682. doi"; pages: " 
            10.1038/ncomms1687"
COMMENT     Sequence Label: pR184
FEATURES             Location/Qualifiers
     source          1..6958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3230..3248
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3251..3269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3271..3289
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3648..3671
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(3695..4315,4316..4702)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MPRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(3768..3787)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(4764..4782)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4974..4993)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4975..4993)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5000..5017)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5000..5016)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5009..5031)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5161..5616
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5248..5267)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5458..5479
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5745..6548)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6549..6769)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6789..6808)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6908..6930
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttt cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggca gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg ggggaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggccgag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctgggtacca
     2281 tcatagagaa atcatatgaa aaatcaaata caactctggg cttggaggaa gtcattataa
     2341 gatccggtta catgggtgtt tgtattgata acattccctc ccaatatagc tcttacaata
     2401 atatgactac attctccaat tcaatttgct atgcaagaaa gaatttaagc tcggttccct
     2461 tatacagaga cttggaaatt caactctcaa atattgcatc ttccagctcc aaaacccaat
     2521 caagcgttgt cttgaacatt ttgaagttag cccaattaac ttcggttaat gttatacatc
     2581 catatgtctt gatggcaact gtaatcctaa caatcctaat gtttctattt attctgtatg
     2641 taactgtccc taagttacca ttcaaactag cggttaataa atttctactg ttgctaagtt
     2701 caactatagt tttgacatgg ggtattggtg cgatgtggac tcatgtaggg ataaatgcaa
     2761 gttacagatt ggttccatca tcaagtatga atataatcac tgtcaagaag ggcaagaagg
     2821 cagcagtaat ggcttggttt agctttgcat tcctcctttt agacagtgtg gttttatggt
     2881 taatattttt aagggatagg aaaagcttga aagatgaaat tgataatgtt ccatgtgccc
     2941 aaaataggta caataactac tcttcggatt catctacatt gcattccaaa gtttagctcg
     3001 agggtcagat tcattatcta caaagactgt aaaaagaaag tggaatattc attcatatca
     3061 tattttttct attaactgcc tggtttcttt taaatttttt attggttgtc gacttgaacg
     3121 gagtgacaat atatatatat atatatttaa taatgacatc attatctgta aatctgattc
     3181 ttaatgctat tctagttatg taagagtggt cccctaggat attgaagtat ccctatcagt
     3241 gatagagaag tccctatcag tgatagagaa tccctatcag tgatagagat gcgtcctcgt
     3301 cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata
     3361 aagattctac aatactagct tttatggtta tgaagaggaa aaattggcag taacctggcc
     3421 ccacaaacct tcaaattaac gaatcaaatt aacaaccata ggatgataat gcgattagtt
     3481 ttttagcctt atttctgggg taattaatca gcgaagcgat gatttttgat ctattaacag
     3541 atatataaat ggaaaagctg cataaccact ttaactaata ctttcaacat tttcagtttg
     3601 tattacttct tattcaaatg tcataaaagt atcaacaaaa aattgttaat atacctctat
     3661 actttaacgt caaggagaaa aaactataag atccatgcct agattagata aaagtaaagt
     3721 gattaacagc gcattagagc tgcttaatgg ggtcggaatc gaaggtttaa caacccgtaa
     3781 actcgcccag aagctaggtg tagagcagcc tacattgtat tggcatgtaa aaaataagcg
     3841 ggctttgctc gacgccttac ccattgagat gttagatagg caccatactc acttttgccc
     3901 tttagaaggg gaaagctggc aagatttttt acgtaataac gctaaaagtt ttagatgtgc
     3961 tttactaagt catcgcgatg gagcaaaagt acatttaggt acacggccta cagaaaaaca
     4021 gtatgaaact ctcgaaaatc aattagcctt tttatgccaa caaggttttt cactagagaa
     4081 tgcattatat gcactcagcg ctgtggggca ctttacttta ggttgcgtat tggaagaaca
     4141 agagcatcaa gtcgctaaag aagaaaggga aacacctact actgatagta tgccgccatt
     4201 attacgacaa gctatcgaat tatttgatcg ccaaggtgca gagccagcct tcttattcgg
     4261 ccttgaattg atcatatgcg gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta
     4321 cagccgcgcg cgtacgaaaa acaattacgg gtctaccatc gagggcctgc tcgatctccc
     4381 ggacgacgac gcccccgaag aggcggggct ggcggctccg cgcctgtcct ttctccccgc
     4441 gggacacacg cgcagactgt cgacggcccc cccgaccgat gtcagcctgg gggacgagct
     4501 ccacttagac ggcgaggacg tggcgatggc gcatgccgac gcgctagacg atttcgatct
     4561 ggacatgttg ggggacgggg attccccggg tccgggattt accccccacg actccgcccc
     4621 ctacggcgct ctggatatgg ccgacttcga gtttgagcag atgtttaccg atgcccttgg
     4681 aattgacgag tacggtgggt agggggcgcg aggatcgtaa taagaattct gtacaaatag
     4741 ggccgcatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
     4801 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     4861 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacgga
     4921 cgcgtgtacg catctagcta gagcggccgc caccgcggtg gagctccaat tcgccctata
     4981 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     5041 gcgttaccca acttaatcgc cttgcagcac atcccccctt cgccagctgg cgtaatagcg
     5101 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcg
     5161 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5221 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5281 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5341 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5401 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5461 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5521 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5581 acgcgaattt taacaaaata ttaacgttta caatttcctg atgcggtatt ttctccttac
     5641 gcatctgtgc ggtatttcac accgcagggt aataactgat ataattaaat tgaagctcta
     5701 atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt ttgctggccg
     5761 catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc taccttagca
     5821 tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc tgtagagacc
     5881 acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca
     5941 ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc tctttgagca
     6001 ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc cttagtatat
     6061 tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag gcctctaggt
     6121 tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg gcccaccaca
     6181 ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc agagtactgc
     6241 aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac
     6301 ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc agtcaagata
     6361 tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa ctccagtaat
     6421 tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc gtgcatgata
     6481 ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt atatgtagct
     6541 ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg ggttaagaat
     6601 actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa tctaagtctg
     6661 tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa atttcaaaga
     6721 aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaattgaaa agcgtggtgc
     6781 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6841 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6901 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
