pORF-CLONE vector DNA酵母表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

pORF-CLONE vector DNA酵母表达载体质粒

The pORF-CLONE vector includes the Cu2+-inducible CUP1 promoter from the yeast
metallothionein gene, which controls expression of genes inserted in the MCS. It also
carries the HIS3 gene, coding imidazol-glycerol-phosphate-dehydrogenase, which enables
the selection of ORFs based on histidine prototrophy. In addition, the vector contains the
E. coli ampicillin resistance (AmpR
) gene for selection in E. coli and the yeast selectable
markers URA3 and LEU2d. For the improved expression of randomly primed cDNAs, a
translation initiation sequence (CAAAATGTCT) that was adapted to S. cerevisiae has
been introduced allowing the translation of cDNAs without their own start codon. For the
detection and purification of expressed gene products, the plasmid includes a tandem
epitope tag positioned N-terminal to the multiple cloning site (MCS), consisting of the
hemagglutinine epitope (HA) and the oligo histidine domain (RGS-His6). The modified
MCS includes a stop codon in each reading frame and allows the directional cloning
(SalI/NotI) of cDNAs. For the cleavage of HIS3p from the fusion protein the recognition
sequence (6P) of the PreScission protease (CTGGAAGTTCTGTTCCAGGGGCCC) has
been additionally inserted. The yeast HIS3 gene was introduced C-terminal to the MCS
(Fig. 2). The translation of the DNA proceeds from the start codon ATG through the tag
sequences into the HIS3 gene. Only if an insert is cloned in the correct reading frame and
if it does not contain any stop codon, a complete fusion protein will be produced (Fig. 2B).
In this case, expression of the gene product leads to histidine prototrophy in a HIS3 S.
cerevisiaestrain. Clones bearing the pORF-CLONE plasmid, but without inserts (Fig. 2A),
remain auxotrophic, as no HIS3 gene is expressed because of translation stops in all three
reading frames. The yeast cells are also expected to be auxotrophic if they contain a
cDNA that is inserted in an incorrect reading frame, leading to internal stop codons and
resulting in the termination of translation before reaching the HIS3 sequence (Fig. 2C, 2D).
In general, mRNA sequences carry many stop codons in reading frames other than the
correct one.
pORF-CLONE can be used for cloning

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