Generation of mMct6 Expression Construct

cDNA encoding for murine Slc16a5 (NM_001080934.1) was purchased from Thermo Fisher Scientific (GeneArt, Rockford, IL, USA). The sequence was designed and optimized to contain 5′ (XmaI) and 3′ (XbaI) restriction sites to flank the open reading frame, a Kozak consensus sequence prior to the start codon, and a C-terminal FLAG tag for immunodetection. Briefly, cDNA was amplified using 5′ and 3′ primers prior to the restriction sites using reverse transcription polymerase chain reaction (RT-PCR) on a BioRad CFX Connect™ RT System. The PCR reaction was purified, and the cDNA and previously used oocyte expression vector (pGH19) [7 (link),34 (link)] were double-digested with XmaI and XbaI. The digested PCR product and linearized vector were further purified and ligated using a T4 DNA ligase reaction mixture. The resulting construct was transformed into chemically competent TOP10 Escherichia Coli cells and the plasmid were isolated, purified, and confirmed via sequencing. The pGH19-mMct6 vector without the FLAG tag was generated using site-directed mutagenesis and used for all activity studies. The pGH19-hMCT6 vector was used and prepared similarly to that described previously [7 (link)].