■ 基本信息
啟動(dòng)子: | URA3 |
復(fù)制子: | pUC |
質(zhì)粒分類: | 酵母系列質(zhì)粒；酵母雜交質(zhì)粒；單雜交類質(zhì)粒 |
質(zhì)粒大小: | 4944bp |
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: | Y1Hgold等酵母菌 |
培養(yǎng)條件: | 30℃，YPDA，有氧 |
5'測(cè)序引物: | pABAI-F（GTTCCTTATATGTAGCTTTCGACA） |
3'測(cè)序引物:
| pABAI-R（CCATCTCGAAAAAGGGTTTGCC）
|
備注: | 低拷貝質(zhì)粒 |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 酵母菌 |
質(zhì)粒用途: | 雜交 |
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
p53-ABAI質(zhì)粒是一種單雜交酵母陽(yáng)性對(duì)照質(zhì)粒。這個(gè)質(zhì)粒具有AbA抗性因而具有很強(qiáng)的篩選能力，可極大地降低背景水平。
p53-AbAi is a yeast reporter vector that serves as a positive control in the Matchmaker Gold Yeast One-Hybrid Library Screening System. The vector contains a p53 binding site, located upstream of the yeast iso-1-cytochrome C minimal promoter and the AUR1-C gene, an antibiotic resistance gene that confers resistance to Aureobasidin A. Expression of AUR1-C, and thus AbA resistance, is induced by the binding of GAL4 activation domain-p53 fusion proteins to the p53 binding site upstream of AUR1-C.
p53-AbAi cannot be propagated episomally in yeast; it can only be stably maintained through integration into the host genome. Integration is accomplished via homologous recombination between the vector’s URA3 gene and the nonfunctional ura3-52 locus of the yeast strain provided in the Matchmaker Gold Yeast One-Hybrid System. URA3 is a nutritional marker that can also be used for the selection of recombinant yeast. To allow propagation and selection in E. coli, the vector also contains a Col E1 origin of replication and an ampicillin resistance gene (Ampr).
p53-AbAi is a positive control reporter vector that is designed to be used in conjunction with the autonomously replicating pGADT7-Rec vector and the p53 control cDNA provided in the Matchmaker Gold Yeast One-Hybrid Library Screening System. To perform control reactions, first linearize the p53-AbAi vector with BstBI, transform the vector into competent yeast cells, and select for integrants on SD/–Ura medium. Next, cotransform the p53 control cDNA and the SmaI-linearized pGADT7-Rec vector (provided) into competent yeast cells, and select for recombinants on SD/–Leu medium containing AbA (see the protocol in the Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual for details).
Transformation of yeast with the linearized p53-AbAi vector will result in the integration of the vector into the yeast chromosome. Subsequent cotransformation of the linearized pGADT7-Rec vector and the p53 control cDNA will yield a construct, through the gap-repair method,that will constitutively express a GAL4 AD-p53 fusion protein. GAL4 AD-p53 will interact with the p53 binding sites on p53-AbAi and stimulate transcription of AUR1-C.
? Suitable host strains: DH5α and other general purpose strains.
? Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.
? E. coli replication origin: ColE1
? Copy number: low
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK970p53-ABAI酵母單雜交質(zhì)粒.txt
