Cloning Strategy

The O. tauri ω3-desaturase full-length sequence was amplified by PCR cycles from cDNA matrix using Q5^® Polymerase by two-step PCR (5′-ATGCGCGCCGCGACGTC-3′ and 5′-CTAGTCGCCCCGCTCCCAGAC-3′), cloned in pGEM^®-T Easy (Promega, Madison, WI, United States) and sequenced (Genwiz, Leipzig, Germany). Amplification from plasmid DNA was achieved with adapted primers to allow further cloning in pOtox-Luc (Moulager et al., 2010) (Restriction sites ApaI, AvrII) and using Gateway^® system according to manufacturer instruction (pDONR 221, pVT102-U-GW for Saccharomyces cerevisiae and pK7W2G2D or pK7YWG2 for N. benthamiana). Primers are provided in Supplementary Tables.

Overexpression in Synechocystis sp. PCC6803 was performed using the pTHT2031S vector after ligation to introduce the synthetic gene using the In-Fusion^® HD cloning kit (Takara Bio, Kusatsu, Japan). Primers are available from the Supplementary Data File.