Figure 17.3.3.

LexA fusion plasmid pEG202. The strong constitutive ADH promoter is used to express bait proteins as fusions to the DNA-binding protein LexA. Restriction sites shown in this map are based on pEG202 sequence data and include selected sites suitable for diagnostic restriction endonuclease digests. A number of restriction sites are available for insertion of coding sequences to produce protein fusions with LexA; the polylinker sequence and reading frame relative to LexA are shown below the map with unique sites marked in bold type. The sequence 5′-CGT CAG CAG AGC TTC ACC ATT G-3′ can be used to design a primer to confirm correct reading frame for LexA fusions. Plasmids contain the HIS3 selectable marker and the 2-μm origin of replication to allow propagation in yeast, and the Ap^r antibiotic resistance gene and the pBR origin of replication to allow propagation in E. coli. In the plasmids pMW101 and pMW103, the ampicillin resistance gene (Ap^r) has been replaced with the chloramphenicol resistance gene (Cm^r) and the kanamycin resistance gene (Km^r), respectively (see Table 17.3.1 for details).