Pcmv gluc 2 control plasmid

First we produced a basic plasmid vector, i.e., a shuttle vector, that contains the following major elements: a gene expression unit driven by the CAG promoter, placed between two INS; a neomycin-resistance gene driven by the human PGK promoter; and a loxP site. A multiple cloning site (MCS) including the BamHI, BsrGI, MluI, and MfeI (MfeI results in ends that are compatiblewith those of EcoRI) restriction sites, in that order, was inserted adjacent to the CAG promoter. The ORF of G-Luc, isolated from the pCMV-G-Luc2 control plasmid (New England Biolabs) by digestion with BamHI and XbaI, was inserted between the BamHI and MfeI sites in the MCS by blunting the XbaI and MfeI ends. The G+KDEL ORF was digested with NheI and EcoRI; the EcoRI site was derived from vector pGEM-T (Easy) (Promega) and inserted into MCS by blunting the NheI end. ORF fragments for the C-WT and the C-Luc variants, including the stop codon, were PCR amplified from the pCMV-C-Luc2 control plasmid (NEB) using a forward primer with a BamHI-BsrGI linker and a reverse primer with an MluI-MfeI linker. Primer sets used in this study are summarized in Table 1.