■ 基本信息
启动子: | GAL1 |
复制子: | 2μFI |
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: | Amp |
真核抗性: | URA3 |
克隆菌株: | DH5a |
培养条件: | 37度 |
表达宿主: | INVSC1等酿酒酵母 |
表达条件: | 30℃，YPD，有氧 |
5'测序引物:
| pYES2-F：GCATAACCACTTTAACTAATAC
|
3'测序引物: | pYES2-R：TCGGTTAGAGCGGATGTG |
■ 质粒属性
质粒宿主: | 酵母菌 |
质粒用途: | 蛋白表达 |
片段类型: |
|
片段物种: |
|
原核抗性: | Amp |
真核抗性: | URA3
|
荧光标记: |
|
■ 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
质粒序列请下载：
ZK949pYES2酿酒酵母质粒.txt
