Primers used for random and site-directed mutagenesis.

a, random mutagenesis; b, site-directed mutagenesis. Underline letters indicate the sequence for ligation-independent cloning; Italic letters indicate the mutation nucleotides. The reverse primers not shown here are reverse and complemented to forward primers. Only forward primers were used for two or more residues replacements according to the instructions of QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent).