LOCUS       sequence-68816-e        6923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rtTA under the control of GAL2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6923)
  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 6923)
  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: pCH044
FEATURES             Location/Qualifiers
     source          1..6923
                     /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"
     CDS             join(3685..4305,4306..4692)
                     /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(3758..3777)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(4733..4751)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4939..4958)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4940..4958)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4965..4982)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4965..4981)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4974..4996)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5126..5581
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5213..5232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5423..5444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5710..6513)
                     /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(6514..6734)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6754..6773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6873..6895
                     /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 ccgcatgcca cgtgatctat attcgaaagg
     3241 ggcggttgcc tcaggaaggc accggcggtc tttcgtccgt gcggagatat ctgcgccgtt
     3301 caggggtcca tgtgccttgg acgatattaa ggcagaaggc agtatcgggg cggatcactc
     3361 cgaaccgaga ttagttaagc ccttcccatc tcaagatggg gagcaaatgg cattatactc
     3421 ctgctagaaa gttaactgtg cacatattct taaattatac aacattctgg agagctattg
     3481 ttcaaaaaac aaacatttcg caggctaaaa tgtggagata ggataagttt tgtagacata
     3541 tataaacaat cagtaattgg attgaaaatt tggtgttgtg aattgctctt cattatgcac
     3601 cttattcaat tatcatcaag aatagtaata gttaagtaaa cacaagatta acataataaa
     3661 aaaaataatt ctttcatagg atccatgcct agattagata aaagtaaagt gattaacagc
     3721 gcattagagc tgcttaatgg ggtcggaatc gaaggtttaa caacccgtaa actcgcccag
     3781 aagctaggtg tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc
     3841 gacgccttac ccattgagat gttagatagg caccatactc acttttgccc tttagaaggg
     3901 gaaagctggc aagatttttt acgtaataac gctaaaagtt ttagatgtgc tttactaagt
     3961 catcgcgatg gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact
     4021 ctcgaaaatc aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat
     4081 gcactcagcg ctgtggggca ctttacttta ggttgcgtat tggaagaaca agagcatcaa
     4141 gtcgctaaag aagaaaggga aacacctact actgatagta tgccgccatt attacgacaa
     4201 gctatcgaat tatttgatcg ccaaggtgca gagccagcct tcttattcgg ccttgaattg
     4261 atcatatgcg gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta cagccgcgcg
     4321 cgtacgaaaa acaattacgg gtctaccatc gagggcctgc tcgatctccc ggacgacgac
     4381 gcccccgaag aggcggggct ggcggctccg cgcctgtcct ttctccccgc gggacacacg
     4441 cgcagactgt cgacggcccc cccgaccgat gtcagcctgg gggacgagct ccacttagac
     4501 ggcgaggacg tggcgatggc gcatgccgac gcgctagacg atttcgatct ggacatgttg
     4561 ggggacgggg attccccggg tccgggattt accccccacg actccgcccc ctacggcgct
     4621 ctggatatgg ccgacttcga gtttgagcag atgtttaccg atgcccttgg aattgacgag
     4681 tacggtgggt aggaattctg tacaaatagg gccgcatcat gtaattagtt atgtcacgct
     4741 tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg
     4801 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat
     4861 ttcaaatttt tctttttttt ctgtacggac gcgtgtacgc atctagagcg gccgccaccg
     4921 cggtggagct ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
     4981 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     5041 cccttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     5101 cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5161 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5221 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5281 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5341 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5401 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5461 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5521 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5581 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc agggtaataa
     5641 ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa
     5701 tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct
     5761 gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat
     5821 aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc
     5881 gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc
     5941 tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc
     6001 aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc
     6061 tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc
     6121 gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat
     6181 tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct
     6241 gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc
     6301 ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc
     6361 taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa
     6421 gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt
     6481 agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt
     6541 tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat
     6601 gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat
     6661 taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa aaaaaaatga
     6721 tgaattgaat tgaaaagcgt ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6781 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     6841 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     6901 accgtcatca ccgaaacgcg cga
//
