Generation of IFI16 Mutant Constructs

IFI16 mutant plasmid constructs were originated from a pCDNA3 human IFI16-HA tagged expression construct kindly provided by Professor Andrew Bowie, Trinity College Dublin. Overlap extension PCR was used to construct a ΔPyrin domain (consisting of amino acids 87–729 of IFI16) or a ΔHIN-A domain+HIN-B domain specific mutations (consisting of amino acids 1–191 and 460–729 of IFI16 and the point mutations K572A, K607A, R611A, S614A, K618A, N654A, K676A, K678A, K703A). Each PCR product was then recloned into a BamHI –and XhoI-digested pCCL-PGK-eGFP (ref. 52 ) together with a PCR-amplified IRES-BFP fragment by NEBuilder HiFi DNA Assembly according to manufactures instructions. For illustration see Fig. 6h.
The mBanana-cGAS fusion construct was engineered by PCR amplification of mBanana and cGAS and subsequent cloning into a NotI-digested pT2/CMV-eGFP.SV40-neo53 (link) by NEBuilder HiFi DNA Assembly according to manufactures instructions.