pHiSi-1酵母单杂交系统载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pHiSi-1酵母单杂交系统载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
pHISi-1 is a yeast integration and reporter vector for use with the MATCHMAKER One-Hybrid System. pHISi-1 contains the yeast HIS3 gene downstream of the MCS and the minimal promoter of the HIS3 locus (PminHIS). Cis-acting sequences of interest (i.e., target elements) can be inserted into the MCS. Without activation by a target element, constitutive HIS3 expression from PminHIS is very low in yeast, but allows enough growth to select for integration when constructing HIS3 reporter strains. During library screening, the leaky expression of HIS3 is controlled by adding 3-AT to the medium. pHISi-1 was constructed by transferring the HIS reporter gene from pHISi to the EcoR I/BamH I sites of pBR322. Leaky HIS3 expression in pHISi-1 is generally lower than that in pHISi, presumably due to differences in the flanking vector sequence.
The yeast HIS3 gene is used as a selectable marker for integration into the nonfunctional his3 locus of the YM4271 host strain after linearizing the vector at the Xho I or Afl II sites. The Kpn I site cannot be used for integration because it cuts within the coding region of the HIS3 gene, and that region is deleted in YM4271. Because it does not carry the URA3 marker, pHISi-1 can be used (together with pLacZi) to construct a dual HIS3/lacZ reporter strain. pHISi-1 cannot replicate autonomously in yeast. pHISi-1 contains a bacterial Col E1 origin (ori) and the ampicillin resistance gene (Ampr ) for propagation and selection in E. coli. Unique restriction sites are in bold.

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