pYES2-CT酵母表达载体说明

pYES2/CT

编号

载体名称

北京华越洋生物VECT2850

pYES2/CT

载体基本信息 出品公司: Invitrogen

载体名称: pYES2-­‐CT, p YES2/CT 质粒类型: 酿酒酵母表达载体 表达水平: 高拷贝 诱导方法: 半乳糖 启动子: GAL1

克隆方法: 多克隆位点，限制性内切酶 载体大小:

5963 b p

5' 测序引物及序列: GAL1-­‐F: A ATATACCTCTATACTTTAACGTC 3' 测序引物及序列: CYC1-­‐R: G CGTGAATGTAAGCGTGAC 载体标签: C-­‐His, C -­‐V5 载体抗性: 氨苄青霉素 筛选标记: URA3报告基因 克隆菌株: TOP10, E .coli

表达菌株: INVSc1, S accharomyces c erevisiae

备注:

酿酒酵母表达载体pYES2/CT 含有URA3报告基因；

GAL1启动子驱动目的基因高水平表达；

半乳糖诱导目基因表达，葡萄糖阻遏目的基因表达，二者起到分子开关作用。 产品目录号: -­‐-­‐

稳定性:

稳表达 组成型/诱导型: 诱导型 病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pYES3-­‐CT多克隆位点

pYES2-­‐CT 载体特征

载体简介

pYES2/CT酿酒酵母表达载体

pYES2/CT载体大小为6.0 k b左右，用于重组蛋白在酿酒酵母细胞中的诱导型表达。诱导剂为半乳糖。重组蛋白的C-­‐端融合了6XHis标签，用于重组蛋白的纯化和检测。

pYES2/CT载体含有以下元件：

1.Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose

and r epression b y g lucose (Giniger e t a l., 1985; W est e t a l., 1984)

2.Multiple cloning site (MCS) with 8 or 9 unique sites (plus two BstX I sites) to facilitate

in-­‐frame c loning w ith t he C-­‐terminal p eptide

3.C-­‐terminal peptide encoding the V5 epitope and a polyhistidine (6xHis) tag for

detection a nd p urification o f y our r ecombinant f usion p rotein

4.2μ origin for episomal maintenance and high copy replication or CEN6/ARSH4

sequence for non-­‐integrative centromeric maintenance and low copy replication (pYC2/CT)

5.URA3 a uxotrophic m arker f or s election o f y east t ransformants

6.Ampicillin r esistance g ene f or s election i n E. c oli

使用pYES2/CT载体表达目的基因的实验流程如下：

1 Consult the multiple cloning site described on page 7 to determine a strategy to clone your gene i n f rame w ith t he C-­‐terminal p eptide.

2 Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on L B p lates c ontaining 50 t o 100 μg/mL a mpicillin.

3 A nalyze y our t ransformants f or t he p resence o f i nsert b y r estriction d igestion.

4 Select a transformant with the correct restriction pattern and sequence to confirm that your gene i s c loned i n f rame w ith t he C-­‐terminal p eptide.

5 Transform your construct into competent INVSc1 cells and select for the appropriate amino acid p rototrophy.

6 T est f or e xpression o f y our r ecombinant p rotein b y w estern b lot a nalysis o r f unctional a ssay.

7 U se m etal-­‐chelating r esin s uch a s P roBond t o p urify y our r ecombinant p rotein.

载体序列

LOCUS pYES2/CT 5963 bp DNA circular SYN

DEFINITION pYES2/CT

ACCESSION

KEYWORDS

SOURCE

ORGANISM other sequences; artificial sequences; vectors.

COMMENT This file is created by Vector NTI

FEATURES Location/Qualifiers

source 1..5963

/organism="pYES2/CT"

/mol_type="other DNA"

promoter 1..451

/label="GAL1_promoter"

misc_feature 414..437

/label="GAL1_primer"

promoter 475..493

/label="T7_promoter"