Figure 17.3.6.

Library plasmid pJG4-5. Library plasmids express cDNAs or other coding sequences inserted into unique EcoRI and XhoI sites as a translational fusion to a cassette consisting of the SV40 nuclear localization sequence (NLS; PPKKKRKVA), the acid blob B42 domain (Ruden et al, 1991), and the hemagglutinin (HA) epitope tag (YPYDVPDYA). Expression of cassette sequences is under the control of the GAL1 galactose-inducible promoter. This map is based on the sequence data available for pJG4-5, and includes selected sites suitable for diagnostic restriction digests (shown in bold). The sequence 5′-CTG AGT GGA GAT GCC TCC-3′ can be used as a primer to identify inserts or to confirm correct reading frame. The pJG4-5 plasmid contains the TRP1 selectable marker and the 2-μm origin to allow propagation in yeast, and the Ap^r antibiotic resistance gene and the pUC origin to allow propagation in E. coli. In the pJG4-5 derivative plasmids pMW104 and pMW102, the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene and kanamycin resistance gene, respectively (see Table 17.3.1 for details). Currently existing libraries are all made in the pJG4-5 plasmid (Gyuris et al., 1993) shown on this figure. Unique sites are marked in bold type.