Vector construction for allotopic expression of Nad9

A nuclear nad9 complementation construct (plasmid pJF1271) was assembled using the GreenGate method⁵³ from plasmids pJF1255, pJF1270, pGGE009, pJF1265 and pGGZ001. The AtUBQ10 (At4g05320) promoter was amplified using primers oJF740 and oJF822 (Supplementary Table 4), the PCR product was cut with Eco31I and ligated into the similarly cut plasmid pGGA000, generating pJF1255. In contrast to pGGA006, the 3′-terminal guanine residue of the putative intron in AtUBQ10 is present immediately upstream of the GoldenGate B-overhang (AACA) in pJF1255. A fusion gene consisting of the sequence encoding the 29-amino-acid N terminus of the Solanum tuberosum formate dehydrogenase (the first 25 of which represent the transit peptide that will be cleaved off after import of the protein into mitochondria²⁴) and the complete nad9 reading frame from the N. tabacum mitochondrial genome (cDNA sequence after RNA editing) was codon optimized for expression in the nucleus of N. tabacum and synthesized as a DNA string (GeneArt). The synthesized DNA was then amplified with primers oJF947 and oJF948 (Supplementary Table 4), digested with HindIII and EcoRI and ligated into the equally cut pGGA000 to create pJF1270. To generate pJF1265 (a GreenGate F-module with a P_nos::hpt::T_nos hygromycin resistance cassette), the hpt coding sequence was amplified with primers oJF937 and oJF938. The backbone of pGGF007 was amplified with primers oJF939 and oJF940 (Supplementary Table 4), the two PCR products were digested with the restriction enzymes KpnI and SacI, and ligated. Plasmid pGGE009 contains the AtUBQ10 (At4g05320) terminator as a GreenGate E-module⁵³. The full sequence of pJF1271 has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"OQ418153","term_id":"2523518527","term_text":"OQ418153"}}OQ418153.