(A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37°C) to degrade template. Vector is digested with XhoI and KpnI, gel purified, then assembled with PCR products using the NEBuilder HiFi DNA Assembly Master Mix (1 hr, 50°C) and transformed into competent cells. Mut_Fwd and Mut_Rvs, mutation-specific primers; Vif_Fwd and Vif_Rvs, common primers; seq, sequencing primer; red circle, site of intended mutation; red cross, intended mutation; red parallel lines, cut sites; orange boxes, overlapping sequences. (B) Overview of flow cytometeric screen. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow cytometry after 36 hr. (C) Illustrative data and gating strategy for flow cytometric screen. A4647 fluorescence indicates abundance of PPP2R5B. For each Vif point mutant, A4647 fluorescence is compared between Green, GFP+, transfected cells (Vif+); grey, GFP-, untransfected cells. (Vif-); dotted line, background staining of control 293Ts (no HA-tagged protein expression). Upper panels, control construct encoding EGFP; lower panels, construct encoding EGFP-P2A-Vif (WT).