Maps of novel vectors for the expression of Type I transmembrane bait and prey proteins in the split-ubiquitin membrane yeast two-hybrid system. (A) The bait vector pCYC-BAIT-Cub-TF is a LEU2-based low copy number (CEN/ARS) vector bearing a weak yeast CYC1 promoter, the MCS, and the Cub domain followed by the TF. The foreign cDNA sequence encoding a transmembrane bait protein of interest is introduced into the MCS in frame to Cub-TF portion. Also shown is the MCS sequence upstream of the Cub-TF fusion containing the unique XbaI, SpeI, PstI, and HindIII restriction sites. (B) The prey vector pADH-PREY-2HA-NubG is a TRP1-based multicopy (2μ) vector bearing a strong yeast ADH1 promoter, the MCS, and two HA tags followed by the NubG domain. The cDNA or a library of genomic or cDNA fragments is fused in frame to the NubG cassette. Also shown is the MCS sequence upstream of the two HA-NubG cassettes containing the unique restriction sites NdeI, NcoI, SmaI, and BamHI. Both bait and prey vectors were constructed as described in the Methods section.
