Schematic diagram of the hGAD65 chloroplast expression vector pXW-GAD-His and the site of integration of the transgene cassette into the chloroplast genome. The hGAD65 expression cassette consists of the C. reinhardtii chloroplast rbcL promoter and 5'UTR upstream of the transgene followed by the rbcL 3'UTR. The transgene cassette was inserted into plasmid vector p322 that contains a cloned 5.7 kb (EcoRI/XhoI fragment) inverted repeat of C. reinhardtii chloroplast DNA, resulting in pXW-GAD-His. The restriction sites used for cloning are indicated. Primers GAD-1/GAD-2 corresponding to the 5' and 3' ends of hGAD65 were used for PCR analysis to confirm the presence of the transgene in transformants, with the expected product size indicated. Primers CP3/CP4 complementary to sequences lying just outside the inverted repeat region of the C. reinhardtii chloroplast DNA were used to determine the site-specific integration of the transgene cassette into the C. reinhardtii chloroplast genome by PCR. The site-specific integration of the transgene cassette was additionally determined by PCR using GAD-1/CP3 and CP4/GAD-2 primer pairs. The regions for homologous recombination are indicated by the crosses. Selection of C. reinhardtii transformants was based on resistance to spectinomycin that was provided by co-transformation with plasmid p228 that contains the 16S rRNA gene conferring spectinomycin resistance .