LOCUS       sequence-90268-e        4867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cassette to replace yeast promoters with an inducible promoter 
            containing 2 lexA boxes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4867)
  AUTHORS   Ottoz DS, Rudolf F, Stelling J
  TITLE     Inducible, tightly regulated and growth condition-independent 
            transcription factor in Saccharomyces cerevisiae.
  JOURNAL   Nucleic Acids Res. 2014 Jul 17. pii: gku616.
  PUBMED    25034689
REFERENCE   2  (bases 1 to 4867)
  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: "Nucleic 
            Acids Res. 2014 Jul 17. pii: gku616."
COMMENT     Sequence Label: FRP1640_PTEF-HygMX-TTEF-insul-(lexA-box)2-Pmin...
FEATURES             Location/Qualifiers
     source          1..4867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            704..2279
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        704..1047
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1048..2076
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2082..2279
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2633..2649)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2634..2650)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2679..2699)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2679..2697)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2718..2734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2718..2734)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2731..2753)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2742..2758
                     /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(2766..2772,2773..2790,2791..2796))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2811..2832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2949..2966)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3120..3708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3200..3219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3879..4670,4671..4739))
                     /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     4502..4521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4740..4844)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcga
      661 tcctcgagag cttgccttgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa
      721 taccctcctt gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta
      781 catttagccc atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac
      841 ggcgcgaagc aaaaattacg gctcctcgct gcggacctgc gagcagggaa acgctcccct
      901 cacagacgcg ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga
      961 tttgccactg aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc
     1021 tcacatcaca tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct
     1081 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     1141 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     1201 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     1261 gcgctcccga ttccggaagt gcttgacatt ggggagttca gcgagagcct gacctattgc
     1321 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     1381 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     1441 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     1501 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     1561 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     1621 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     1681 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     1741 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     1801 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     1861 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     1921 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     1981 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     2041 cgacgcccca gcactcgtcc gagggcaaag gaataatcag tactgacaat aaaaagattc
     2101 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
     2161 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
     2221 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
     2281 tgtcgattcg atactaacgc cgccatccag tgtttaaacg aactagtgcg gccgcaataa
     2341 tatataaacc tgtataatat aaccttgaag actatatttc ttttcgagct ccctaggtgc
     2401 tgtatatact cacagcataa ctgtatatac acccagggtc taggtgctgt atatactcac
     2461 agcataactg tatatacacc cagggtctag agcatgtgct ctgtatgtat ataaaactct
     2521 tgttttcttc ttttctcttc tttccttata cattaggacc tttgcagcat aaattactat
     2581 acttctatac tagtggatcc cccgggctgc aggaattcga tatcaagctt atcgataccg
     2641 tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg
     2701 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2761 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2821 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2881 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2941 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3001 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3061 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3121 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3181 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3241 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3301 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3361 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3421 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3481 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3541 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3601 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3661 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3721 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3781 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3841 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3901 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3961 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4021 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4081 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4141 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4201 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4261 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4321 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4381 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4441 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4501 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4561 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4621 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4681 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4741 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4801 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4861 gtgccac
//
