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
pYES2酿酒酵母质粒使用说明:
1、收到质粒干粉后请先5000rpm离心1min,再加入20μl无菌水溶解质粒,室温放置1min;
2、从-80℃冰箱中取出相应的感受态,置于冰盒上解冻,并做好标记;
3、取2μl质粒加至100μl感受态中,冰浴30min;
4、42℃热激90s,再冰浴2min;
5、加入900μl无抗的LB液体培养基,180rpm震荡培养45min;
6、6000rpm离心5min,仅留100ul上清混匀菌体沉淀;
7、混匀后的菌液加至对应抗性的LB平板上,倒入适量玻璃珠,涂匀液体;
8、将平板正向培养1h,再倒置培养12h~16h;
9、挑取单克隆菌落至对应抗性的LB液体培养基中,震荡培养12h~16h,根据实验需要提取质粒。
pYES2酿酒酵母质粒注意事项:
1、如果您收到的是甘油菌种,请先四区划线,挑取单克隆培养。
2、如果第二天转化平板长的过多,请将质粒按比例稀释后再转化。
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