LOCUS       Exported                4961 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Cassette to replace yeast promoters with an inducible promoter 
            containing 4 lexA boxes.
ACCESSION   .
VERSION     .
KEYWORDS    FRP1642_PTEF-HygMX-TTEF-insul-(lexA-box)4-PminCYC1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4961)
  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 4961)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4961
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..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
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /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(2727..2743)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2728..2744)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2773..2793)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2773..2791)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2812..2828)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2812..2828)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2825..2847)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2836..2852
                     /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(2860..2890)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2905..2926
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3043..3060)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3214..3802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3294..3313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3973..4833)
                     /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     4596..4615
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4834..4938)
                     /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 gtgctgtata tactcacagc ataactgtat
     2521 atacacccag ggtctaggtg ctgtatatac tcacagcata actgtatata cacccagggt
     2581 ctagagcatg tgctctgtat gtatataaaa ctcttgtttt cttcttttct cttctttcct
     2641 tatacattag gacctttgca gcataaatta ctatacttct atactagtgg atcccccggg
     2701 ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc
     2761 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     2821 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2881 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2941 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3001 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3061 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3121 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3181 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3241 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3301 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3361 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3421 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3481 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3541 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3601 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     3661 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3721 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3781 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3841 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3901 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3961 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4021 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4081 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4141 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4201 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4261 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4321 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4381 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4441 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4501 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4561 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4621 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4681 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4741 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4801 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4861 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4921 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
