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For the cholesterol sensor strains generation the PFO-derived D4 domain mutants YDA (D434W Y415A A463W) and YQDA (D434W Y415A A463W Q433W) fused to a mCherry N-terminal tag were cloned using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520) and introduced into pBlueScriptII with a VHA-6 promoter and tub terminator using the primers pvha6_fwd and pvha6_rev for the promoter amplification, for the sensor amplification D4H_fwd and D4H_rev and tubter_fwd and tubter_rev for tubulin terminator were used (Table S1)