Generating Missense Mutations in ABCA4

Missense mutations in ABCA4-1D4 were generated by PCR based site-directed mutagenesis as previously described^{21 ,22 (link)} Briefly, mutagenesis was performed using a cloning cassette encoding F213 (Afe1 restriction site) to R943 (Fse1 restriction site) in pcDNA3. PCR was performed with Q5 mutagenesis kit (New England Biolabs) with overlapping primers (Supplementary Table 2) as per manufacturer’s recommendations. Each amplified ABCA4 F213-R943 cDNA was sequenced before cloning back into pCEP4 plasmid containing ABCA4-1D4 tag that had been cut with restriction enzymes Afe1 and Fse1. All DNA constructs were verified by Sanger sequencing.