Sau3ai fd

gDNA was extracted as described and partially digested with Sau3AI FD (Thermo Fisher Scientific). Sau3AI FD was diluted 300-fold in 1× FastDigest buffer (Thermo Fisher Scientific) and was applied to the reaction mixture for a 3,000-fold enzyme dilution. Digested DNA was size-separated via agarose gel electrophoresis and fragments of ∼10 kbp were extracted and purified using Zymoclean Large Fragment DNA Recovery Kit, subcloned in BamHI-digested pRU1097, and electroporated in E. coli K12 DH10B MegaX. Plasmid DNA of ∼14,000 E. coli transformant colonies was extracted, representing more than 99.99% theoretical coverage of the PfAR-1 genome.