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.