Figure 1. Cloning different subunits forP. pastoris expression of multisubunit membrane complex in-frame with fluorescent proteins.

Shown are the vectors containing mVenus and His₁₀-tag (left) and mCerulean including a strepII-tag (right) used to clone different subunits for co-expression in P. pastoris. Salient features are: H, His₁₀-tag; S, strepII-tag; TEV, TEV protease cleavage sequence; mVenus and mCerulean, the fluorescent proteins. Upper parts show a close-up of the region between the AOX1 promoter (green) and the TEV sequence (white, start of the TEV sequence is given in bold letters) after digestion with BsmBI and T4 DNA polymerase. Also shown is a theoretical PCR product (any gene of interest) after T4 DNA polymerase treatment displaying ends complementary to the vector.