■ 基本信息
啟動(dòng)子: | GAL1 |
復(fù)制子: | 2μFI |
終止子: | CYC1 terminator |
質(zhì)粒分類: | 酵母系列質(zhì)粒；酵母表達(dá)質(zhì)粒；釀酒酵母質(zhì)粒 |
質(zhì)粒大小: | 5856bp |
原核抗性: | Amp |
真核抗性: | URA3 |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37度 |
表達(dá)宿主: | INVSC1等釀酒酵母 |
表達(dá)條件: | 30℃，YPD，有氧 |
5'測(cè)序引物:
| pYES2-F：GCATAACCACTTTAACTAATAC
|
3'測(cè)序引物: | pYES2-R：TCGGTTAGAGCGGATGTG |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 酵母菌 |
質(zhì)粒用途: | 蛋白表達(dá) |
片段類型: |
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片段物種: |
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原核抗性: | Amp |
真核抗性: | URA3
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熒光標(biāo)記: |
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■ 質(zhì)粒簡(jiǎn)介
pYES2的是一個(gè)5.9 kb的載體，設(shè)計(jì)用來(lái)在釀酒酵母（Saccharomyces cerevisiae）中誘導(dǎo)表達(dá)重組蛋白。載體的特點(diǎn)在于基因插入載體的構(gòu)建簡(jiǎn)單，以及能夠使用原養(yǎng)型尿嘧啶進(jìn)行轉(zhuǎn)化株的篩選。
1. 酵母GAL1啟動(dòng)子，能夠在釀酒酵母中被半乳糖高水平的誘導(dǎo)蛋白表達(dá)目的蛋白，同時(shí)能夠被葡萄糖抑制表達(dá)；
2.多克隆位點(diǎn)可以使用的很多限制酶切位點(diǎn)，便于基因插入；
3.CYC1終止子能夠有效終止mRNA的轉(zhuǎn)錄；
4.能夠利用URA3基因篩選帶有ura3基因型的酵母宿主菌株轉(zhuǎn)化子；
5.氨芐抗性基因能夠方便在大腸桿菌中的進(jìn)行載體篩選。
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.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK949pYES2釀酒酵母質(zhì)粒.txt
