Plasmid Construction for Mitochondrial Studies

Universal human reference RNA (Thermo Fisher Scientific, QS0639) was used to generate cDNA with the Thermo Scientific™ RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), from which coding sequences of genes and sorting signals of OPA1(1-209) and MICU1(1-60)⁵³ were amplified by PCR. DELE1 and DELE1(ΔMTS) have been described before^{11 (link)} and variants of DELE1 were generated from the DELE1 coding sequence by PCR. The following sequences were derived from plasmids obtained from Addgene: Su9(1-69) (#23214), TEV protease (#116062), PINK1 (#13320). Sequences for CLUH, COX8A(1-36), 3×ALFA-tag, split mNG2, scHB, scCOX5A(1-118), scPSD1(1-108) and 3C protease were codon-optimized and obtained by gene synthesis (IDT; Thermo Fisher Scientific). Restriction digest and ligation into destination vectors were performed following standard procedures. Cloned constructs were verified by Sanger sequencing.