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启动子 | URA3 |
复制子 | pUC |
质粒分类 | 酵母系列质粒;酵母杂交质粒;单杂交类质粒 |
质粒大小 | 4944bp |
原核抗性 | Amp |
真核抗性 | URA3 |
克隆菌株 | DH5α |
培养条件 | 37℃ |
表达宿主 | Y1Hgold等酵母菌 |
培养条件 | 30℃,YPDA,有氧 |
5'测序引物 | pABAI-F(GTTCCTTATATGTAGCTTTCGACA) |
3'测序引物 | pABAI-R(CCATCTCGAAAAAGGGTTTGCC) |
备注 | 低拷贝质粒 |
质粒宿主 | 酵母菌 |
质粒用途 | 杂交 |
原核抗性 | 氨苄青霉素 |
真核抗性 | URA3 |
p53-ABAI质粒是一种单杂交酵母阳性对照质粒。这个质粒具有AbA抗性因而具有很强的筛选能力,可极大地降低背景水平。
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
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