LOCUS       40924_40102        7486 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Recombinase expression vector pSH68, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7486)
  AUTHORS   Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH.
  TITLE     A new efficient gene disruption cassette for repeated use in budding
            yeast
  JOURNAL   Nucleic Acids Res. 24 (13), 2519-2524 (1996)
  PUBMED    8692690
REFERENCE   2  (bases 1 to 7486)
  AUTHORS   Hegemann JH, Heick SB.
  TITLE     Delete and Repeat: A Comprehensive Toolkit for Sequential Gene 
            Knockout in the Budding Yeast Saccharomyces cerevisiae
  JOURNAL   Methods Mol. Biol. 765, 189-206 (2011)
  PUBMED    21815094
REFERENCE   3  (bases 1 to 7486)
  AUTHORS   Hegemann JH, Heick SB.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-OCT-2010) Lehrstuhl fur Funktionelle Genomforschung 
            der Mikroorganismen, Heinrich-Heine-Universitaet, 
            Universitatsstrasse 1, Duesseldorf, NRW 40225, Germany
REFERENCE   4  (bases 1 to 7486)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7486)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "1996"; volume: "24"; issue: "13"; pages: 
            "2519-2524"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Methods 
            Mol. Biol. 765, 189-206 (2011)"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (13-OCT-2010) Lehrstuhl fur Funktionelle Genomforschung der 
            Mikroorganismen, Heinrich-Heine-Universitaet, Universitatsstrasse 1,
            Duesseldorf, NRW 40225, Germany"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        317..503
                     /label=HIS3 promoter
     CDS             504..1595
                     /codon_start=1
                     /label=LEU2
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     rep_origin      complement(1858..2313)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2458..2474
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2484..2502
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2521..2768)
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     CDS             complement(3090..4118)
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRTLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(4216..4232)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(4247..4688)
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     promoter        complement(4712..4730)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4751..4767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4775..4791)
                     /label=lac operator
                     /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(4799..4829)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4844..4865)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5153..5741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5915..6772)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6773..6877)
                     /label=AmpR promoter
     misc_feature    6914..7417
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgcgtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata
      421 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc
      481 aggcaagata aacgaaggca aagatgtctg cccctaagaa gatcgtcgtt ttgccaggtg
      541 accacgttgg tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg
      601 ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgacg
      661 ctacaggtgt cccacttcca gatgaggcgc tggaagcctc caagaaggtt gatgccgttt
      721 tgttaggtgc tgtgggtggt cctaaatggg gtacaggtag tgttagacct gaacaaggtt
      781 tactaaaaat ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat
      841 ccgactctct tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg
      901 ttgttgtcag agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg
      961 atggtgtcgc ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa
     1021 tggccgcttt catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag
     1081 ctaatgtttt ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg
     1141 aatttcctac attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta
     1201 agaacccaac ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct
     1261 ccgatgaagc ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct
     1321 ctttgccaga caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag
     1381 atttgccaaa gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga
     1441 aattgtcatt gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt
     1501 tggatgcagg tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg
     1561 atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaatg acaccgatta tttaaagctg
     1621 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta
     1681 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg
     1741 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac
     1801 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     1861 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1921 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     1981 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2041 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2101 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2161 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2221 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2281 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca
     2341 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     2401 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     2461 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccggcc
     2521 gcaaattaaa gccttcgagc gtcccaaaac cttctcaagc aaggttttca gtataatgtt
     2581 acatgcgtac acgcgtctgt acagaaaaaa aagaaaaatt tgaaatataa ataacgttct
     2641 taatactaac ataactataa aaaaataaat agggacctag acttcaggtt gtctaactcc
     2701 ttccttttcg gttagagcgg atgtgggggg agggcgtgaa tgtaagcgtg acataactaa
     2761 ttacatgact cgaggtcgac aattccaacc ttacccaaga gttcgccaaa ctcagacatc
     2821 actttangca aaaccgcgcc gtgcttcttc ctcggtggca ttcatcacga aatgttcagc
     2881 actacgcata cttttgacag gaaacgcaac ggatattgag tcaatatcag gcattctatc
     2941 gctcagcttt acagtgacaa tgacggctgg cgactgaata ttagtgctta cagacagcac
     3001 tacatatttt ccgtcgatgt tgaaatcctt tctcatatgt caccataaat atcaaataat
     3061 tatagcaatc atttacgcgt taatggctaa tcgccatctt ccagcaggcg caccattgcc
     3121 cctgtttcac tatccagggt acggatatag ttcatgacaa tatttacatt ggtccagcca
     3181 ccagcttgca tgatctccgg tattgaaact ccagcgcggg ccatatctcg cgcggctccg
     3241 acacgggcac tgtgtccaga ccaggccagg tatctctgac cagagtcatc cttagcgccg
     3301 taaatcaatc gatgagttgc ttcaaaaatc ccttccaggg cgcgagttga tagctggctg
     3361 gtggcagatg gcgcggcaac accatttttt ctgacccggc aaaacaggta gttattcgga
     3421 tcatcagcta caccagagac ggaaatccat cgctcgacca gtttagttac ccccaggcta
     3481 agtgccttct ctacacctgc ggtgctaacc agcgttttcg ttctgccaat atggattaac
     3541 attctcccac cgtcagtacg tgagatatct ttaaccctga tcctggcaat ttcggctata
     3601 cgtaacaggg tgttataagc aatccccaga aatgccagat tacgtatatc ctggcagcga
     3661 tcgctatttt ccatgagtga acgaacctgg tcgaaatcag tgcgttcgaa cgctagagcc
     3721 tgttttgcac gttcaccggc atcaacgttt tcttttcgga tccgccgcat aaccagtgaa
     3781 acagcattgc tgtcacttgg tcgtggcagc ccggaccgac gatgaagcat gtttagctgg
     3841 cccaaatgtt gctggatagt ttttactgcc agaccgcgcg cctgaagata tagaagataa
     3901 tcgcgaacat cttcaggttc tgcgggaaac catttccggt tattcaactt gcaccatgcc
     3961 gcccacgacc ggcaaacgga cagaagcatt ttccaggtat gctcagaaaa cgcctggcga
     4021 tccctgaaca tgtccatcag gttcttgcga acctcatcac tcgttgcatc gaccggtaat
     4081 gcaggcaaat tttggtgtac ggtcagtaaa ttggacattt aacactcaga taatggtttt
     4141 aagtaaagtg tacaggatcg gctctgcccc tcgacggtat cgataagctt gatatcgaat
     4201 tcctgcagcc cgggggatcc actagttcta gaatccgggg ttttttctcc ttgacgttaa
     4261 agtatagagg tatattaaca attttttgtt gatactttta ttacatttga ataagaagta
     4321 atacaaaccg aaaatgttga aagtattagt taaagtggtt atgcagtttt tgcatttata
     4381 tatctgttaa tagatcaaaa atcatcgctt cgctgattaa ttaccccaga aataaggcta
     4441 aaaaactaat cgcattatca tcctatggtt gttaatttga ttcgttcatt tgaaggtttg
     4501 tggggccagg ttactgccaa tttttcctct tcataaccat aaaagctagt attgtagaat
     4561 ctttattgtt cggagcagtg cggcgcgagg cacatctgcg tttcaggaac gcgaccggtg
     4621 aagacgagga cgcacggagg agagtcttcc ttcggagggc tgtcacccgc tcggcggctt
     4681 ctaatccgta ctagagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     4741 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4801 acataggagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg aggtaactca
     4861 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4921 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4981 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5041 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5101 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5161 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5221 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5281 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5341 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5401 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5461 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5521 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5581 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5641 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5701 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5761 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5821 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5881 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5941 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6001 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6061 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6121 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6181 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6241 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6301 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6361 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6421 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6481 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6541 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6601 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6661 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6721 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6781 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6841 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6901 ctgggtcctt ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat
     6961 atataaatta aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc
     7021 gaagatgtaa aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc
     7081 tgctctcagg tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa
     7141 aatcctgtga ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt
     7201 ttcttgtcta ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg
     7261 tttatttttt tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc
     7321 aaatacaaaa cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa
     7381 aagatacgag gcgcgtgtaa gttacaggca agcgatccgt cctaagaaac cattattatc
     7441 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
