pYES2酿酒酵母质粒

基本信息

启动子:	GAL1 promoter
复制子:	2μ ori,ori,FI ori
终止子:	CYC1 terminator
质粒分类:	酵母系列质粒；酵母表达质粒；酿酒酵母质粒
质粒大小:	5856bp
原核抗性:	氨苄青霉素Amp（100μg/ml）
筛选标记:	URA3
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，LB，有氧
表达宿主:	INVSC1等酿酒酵母
培养条件:	30℃，YPD，有氧
5'测序引物:	T7(TAATACGACTCACTATAGGG)
3'测序引物:	CYC1-Ter (GTGACATAACTAATTACATGATG)

质粒简介

         pYES2的是一个5.9 kb的载体，设计用来在酿酒酵母（Saccharomyces cerevisiae）中诱导表达重组蛋白。载体的特点在于基因插入载体的构建简单，以及能够使用原养型尿嘧啶进行转化株的筛选。
         1. 酵母GAL1启动子，能够在酿酒酵母中被半乳糖高水平的诱导蛋白表达目的蛋白，同时能够被葡萄糖抑制表达；
         2.多克隆位点可以使用的很多限制酶切位点，便于基因插入；
         3.CYC1终止子能够有效终止mRNA的转录；
         4.能够利用URA3基因筛选带有ura3基因型的酵母宿主菌株转化子；
         5.氨苄抗性基因能够方便在大肠杆菌中的进行载体筛选。
         pYES2 is 5.9 kb vector respectively, designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae. Features of the vectors allow purification and detection of expressed proteins (see pages 13–20 for more information). The vectors contain the following elements: Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose (Giniger et al., 1985; West et al., 1984) Multiple cloning site (MCS) with 8 or 9 unique sites (plus two BstX I sites) to facilitate in-frame cloning with the C-terminal peptide C-terminal peptide encoding the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of your recombinant fusion protein 2μorigin for episomal maintenance and high copy replication (pYES2/CT and pYES3/CT) or CEN6/ARSH4 sequence for non-integrative centromeric maintenance and low copy replication (pYC2/CT) URA3 or TRP1 auxotrophic marker for selection of yeast transformants Ampicillin resistance gene for selection in E. coli.
         Use the following outline to clone and express your gene of interest in pYES2.
         • Consult the multiple cloning site described on page 3 to design a strategy to clone your gene in pYES2.
         • Ligate your insert into pYES2 and transform into E. coli. Select transformants on LB plates containing 50 to 100 µg/ml ampicillin.
         • Analyze your transformants for the presence of insert by restriction digestion.
         • Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation.
         • Transform your construct into competent INVSc1 cells and select for uracil prototrophy.
         • Test for expression of your recombinant gene by Western blot analysis or functional assay.

质粒图谱

质粒序列

LOCUS       Exported                5856 bp ds-DNA     circular SYN 07-FEB-2013
DEFINITION  High-copy episomal vector for galactose-inducible expression of 
            proteins in Saccharomyces cerevisiae.
KEYWORDS    pYES2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5856)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
  JOURNAL   Exported Sunday, September 11, 2016 from Plasmid
FEATURES             Location/Qualifiers
     source          1..5856
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     promoter        2..443
                     /gene="S. cerevisiae GAL1"
                     /note="GAL1 promoter"
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2..119
                     /bound_moiety="Gal4"
                     /note="UAS"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     promoter        475..493
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    501..600
                     /note="MCS"
                     /note="multiple cloning site"
     terminator      609..856
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     rep_origin      complement(1097..1685)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1856..2716)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance fo ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             complement(2812..3615)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /note="URA3"
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3616..3831)
                     /gene="S. cerevisiae URA3"
                     /note="URA3 promoter"
     rep_origin      4435..5315
                     /note="2u ori"
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(5384..5839)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt
       61 cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga
      121 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
      181 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga
      241 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat
      301 taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc
      361 ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac
      421 ctctatactt taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac
      481 gactcactat agggaatatt aagcttggta ccgagctcgg atccactagt aacggccgcc
      541 agtgtgctgg aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
      601 gggccgcatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc
      661 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
      721 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag
      781 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
      841 gaaggcttta atttgcggcc ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      901 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      961 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1021 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa gcccaggaac cgtaaaaagg
     1081 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1141 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1201 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1261 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1321 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1381 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1441 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1501 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     1561 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1621 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1681 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1741 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1801 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1861 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1921 cctgactccc cgtcgtgtag ataactacga tacgggagcg cttaccatct ggccccagtg
     1981 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2041 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2101 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2161 ttggcattgc tacaggcatc gtggtgtcac tctcgtcgtt tggtatggct tcattcagct
     2221 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2281 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2341 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2401 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2461 gcccggcgtc aatacgggat aatagtgtat cacatagcag aactttaaaa gtgctcatca
     2521 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2581 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2641 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2701 aatgttgaat actcatactc ttcctttttc aatgggtaat aactgatata attaaattga
     2761 agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg
     2821 ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac
     2881 cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt
     2941 agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa
     3001 acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct
     3061 ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt
     3121 agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc
     3181 tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc
     3241 caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga
     3301 gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa
     3361 attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt
     3421 caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc
     3481 cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg
     3541 catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata
     3601 tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt
     3661 taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct
     3721 aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt
     3781 tcaaagaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga attgaaaagc
     3841 tagcttatcg atgataagct gtcaaagatg agaattaatt ccacggacta tagactatac
     3901 tagatactcc gtctactgta cgatacactt ccgctcaggt ccttgtcctt taacgaggcc
     3961 ttaccactct tttgttactc tattgatcca gctcagcaaa ggcagtgtga tctaagattc
     4021 tatcttcgcg atgtagtaaa actagctaga ccgagaaaga gactagaaat gcaaaaggca
     4081 cttctacaat ggctgccatc attattatcc gatgtgacgc tgcagcttct caatgatatt
     4141 cgaatacgct ttgaggagat acagcctaat atccgacaaa ctgttttaca gatttacgat
     4201 cgtacttgtt acccatcatt gaattttgaa catccgaacc tgggagtttt ccctgaaaca
     4261 gatagtatat ttgaacctgt ataataatat atagtctagc gctttacgga agacaatgta
     4321 tgtatttcgg ttcctggaga aactattgca tctattgcat aggtaatctt gcacgtcgca
     4381 tccccggttc attttctgcg tttccatctt gcacttcaat agcatatctt tgttaacgaa
     4441 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     4501 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa
     4561 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgacgaga gcgctaattt
     4621 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     4681 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     4741 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     4801 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     4861 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     4921 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     4981 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     5041 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     5101 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     5161 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     5221 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     5281 ttttgagcaa tgtttgtgga agcggtattc gcaatgggaa gctccacccc ggttgataat
     5341 cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat
     5401 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa cgaatagccc
     5461 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5521 ccagtttcca acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5581 agggtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5641 tcgaggtgcc gtaaagcagt aaatcggaag ggtaaacgga tgcccccatt tagagcttga
     5701 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggggct
     5761 agggcggtgg gaagtgtagg ggtcacgctg ggcgtaacca ccacacccgc cgcgcttaat
     5821 ggggcgctac agggcgcgtg gggatgatcc actagt