Fluorescent Protein Expression Cassette

The hygromycin resistance cassette from pSELECT100 [19 (link)] and a gateway cloning site with luciferase reporter [49 (link)] were transferred to the high-copy pGEM backbone to form pNOC-Dlux. The LDSP 3′ UTR and terminator was amplified by PCR on Nannochloropsis genomic DNA, using primers given in Additional file 14: Table S4. The PCR product was blunt cloned with Zero Blunt^® PCR Cloning Kit (Invitrogen, ThermoFisher Scientific), sequenced and transferred to the SacI and AflII sites in the pNoc-Dlux plasmid. The elongation factor (EF) promoter was amplified by PCR from Nannochloropsis genomic DNA (primers given in Additional file 14: Table S4) and inserted in the pENTR gateway entry vector by using pENTR™/D-TOPO^® Cloning Kit (Invitrogen, ThermoFisher Scientific), sequenced, and transferred to pNoc-Dlux-LDSP terminator by a LR clonase reaction (Invitrogen). The luciferase reporter was removed by digestion with AscI and SacI and replaced with venus fluorescent protein (Additional file 9: Figure S8A) or green fluorescent protein (Additional file 9: Figure S8B) genes, amplified by PCR with the primers given in Additional file 14: Table S4, blunt cloned as described above, sequenced and inserted into the HpaI and MluI sites.