■ 基本信息
啟動(dòng)子: | URA3 |
復(fù)制子: | ColE1 ori |
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: | 4894bp |
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
pABAI質(zhì)粒是一種單雜交酵母報(bào)告質(zhì)粒，可以識(shí)別和分析蛋白質(zhì)與DNA之間的相互作用，而不僅僅是證明蛋白之間的相互作用。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。
pABAI is a yeast reporter vector that can be used in one-hybrid assays to identify and characterize DNA-binding proteins. The vector, which was specifically designed for use with the Matchmaker Gold Yeast One-Hybrid Library Screening System, contains a multiple cloning site upstream of the yeast iso-1-cytochrome C minimal promoter(not shown) and the AUR1-C gene, an antibiotic resistance gene that confers resistance to Aureobasidin A. A cis-acting element (i.e., a target sequence) can be inserted into the MCS and used as bait to screen GAL4 AD/cDNA fusion libraries for proteins that interact with the target sequence. Positive protein-DNA (i.e.,one-hybrid) interactions drive the expression of AUR1-C. As a result, one-hybrid interactions can be detected by selecting for yeast that are resistant to AbA.
pABAI cannot be propagated episomally in yeast; it can only be stablymaintained through integration into the host genome. Integration is accomplished via homologous recombination between the vector’s URA3 gene and the ura3-52 locus of the yeast strain provided in theMatchmaker Gold Yeast One-Hybrid System. URA3 is a nutritional marker that can also be used for selection of recombinant yeast. The vector also contains a Col E1 origin of replication and an ampicillin resistance gene (Ampr) for propagation and selection in E.coli.
To use pAbAi in a one-hybrid assay, clone one or more copies of a cis-acting element into the MCS. To facilitate recombination of the vector with the host’s ura3-52 locus, linearize the vector with either BbsI or BstBI, then introduce the linearized vector into competent yeast cells using the protocols in the Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual.
Insertion of your target sequence may alter the basal expression of AUR1-C. Therefore, before starting a onehybrid analysis, basal levels of AUR1-C should be determined as described in the User Manual.
? Suitable host strains: DH5α and other general purpose strains.
? Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.
? Copy number: low
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK969pABAI酵母單雜交質(zhì)粒.txt
