Engineered CHIKV-GFP Mutants

Using the IVA cloning approach [46 (link)], we simultaneously introduced two mutations (nsP2 S259P and R650D) into the CHIKV-GFP. Briefly, mutagenic primers were designed according to the IVA protocol, synthesized by Integrated DNA Technologies (IDT), and then used for mutagenic PCR with Q5 2× Master Mix (New England Biolabs #M0492S) instead of the Phusion polymerase. Following DpnI digestion (Thermo Fisher Scientific #FD1703) to remove residual wild-type plasmid, the PCR was transformed into Turbo competent cells (New England Biolabs #C2984I) and incubated at 30°C overnight. The following day, individual colonies were grown in 2× YT media and mini-preps were subsequently performed (Macherey Nagel #740588). The presence of mutations was confirmed using Sanger sequencing.