Piresneo3 mammalian expression vector

The human PIS gene (GenBank: NM_006319), the human CDS1 gene (GenBank: NM_001263) and the human CDS2 gene (GenBank: NM_003818) were obtained from Kazusa DNA Research Institute (Kisarazu, Japan). An oligonucleotide encoding the FLAG (DYKDDDDK) epitope was appended to the 5′ end of the genes via PCR. Plasmids for FLAG-PIS, FLAG-CDS1 or FLAG-CDS2 expression were constructed by inserting each PCR product into the pIRESneo3 mammalian expression vector (Clontech, Mountain View, CA, USA), which promotes the establishment of pools of stably transfected cells^{51 (link)}.

Using PCR, an oligonucleotide encoding a FLAG (DYKDDDDK)-tagged epitope was appended to the 5’ end of the human PGS1 gene (GenBank: NM_024419). These PCR products were ligated into the Nhe I and Eco RI sites of the pIRESneo3 mammalian expression vector (Clontech, Mountain View, CA, USA) to generate the plasmids, pIRESneo3/FLAG-PGS1. pIRESneo3 contains the internal ribosome entry site, which permits the translation of two open reading frames from one messenger RNA. This expression system facilitates the establishment of pools of stably transfected cell lines whereby nearly all cells surviving in selective media express the gene of interest, since the neomycin phosphotransferase gene is expressed under the control of the same promoter57 (link).