Pcdna3 ha

For the generation of dual luciferase expression constructs, overlapping oligonucleotide pairs (Integrated DNA Technologies (IDT)) containing sequence flanking the stop codons (6 nt 5′ and 12 nt 3′) (see Table S2) of the predicted readthrough candidates were annealed and ligated with PspXI/BglII-digested pSGDluc (13 (link)).
The HA-VDR-TGA expression clone was made by PCR amplifying the VDR coding sequence plus 200 nucleotides of 3′UTR from HEK293T cDNA and then cloned BamHI/XbaI in-frame with the influenza HA tag in pcDNA3-HA (Invitrogen). HA-VDR-TGG and HA-VDR-TAATAA were generated by two-step PCR mutagenesis using HA-VDR-TGA as template (see Table S2 for PCR primers). RXRα was synthesized by IDT as a G Block and digested with incorporated 5′ BglII and 3′ XbaI restriction sites then ligated with BamHI/XbaI-digested pcDNA3-HA to generate HA-RXRα. GFP-VDR fusion constructs were made by subcloning the VDR-TGA, VDR-TGG, and VDR-TAATAA cassettes from the pcDNA3-HA constructs just described into pEGFP-C3 (Clontech).
Wildtype (WT) and mutant VDRE–firefly luciferase fusions were generated by restricting WT and mutant G Blocks (IDT) (see Table S2 for sequences) with SacI/BglII then ligating with SacI/BglII-restricted pDLuc (75 (link)). SacI/BglII digestion of pDLuc removes the SV40 promoter and Renilla luciferase. All constructs were verified by DNA sequencing.