Fig. 2. Chloroplast transformation of C. reinhardtii with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the chloroplast ATP synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S).