Plasmid construction
All plasmids were constructed using Start-Stop assembly[15]. This is a level-based cloning system with basic genetic elements as discrete standardised ‘level 0’ parts. The type IIS restriction enzyme SapI was used to assemble transcription units (level 1) from the level 0 parts and then these were combined, along with flanking arms for homologous recombination, using BsaI to create the final level 2 plasmids. Some minor modifications were made to the Start-Stop acceptor vector, which are detailed inSupplementary File S1 along with the basic assembly strategy for the level 2 constructs. The coding sequence for the mVenus.ME variant carrying a Q69M change[34] was codon optimized for the C. reinhardtii chloroplast, ordered as a synthetic gene termed mVenCP (GeneArt; Thermo Fisher Scientific), and cloned into a level 0 acceptor vector. See Supplementary File S2 for GenBank format vector maps of all level 2 constructs assembled in this study.