Figure 3

Adaptation of existing Gateway Destination vectors for the expression of fusion proteins. A. Construction of adapter vector pDONR221-R4-R3. A PCR product corresponding to the attR4-attR3-containing Gateway cassette in pDEST-R4-R3 was amplified with primers that provided flanking attB1 and attB2 sites. An inactivating mutation was introduced into the CAT gene (red X) in pDONR221 by site-directed mutagenesis so that ccdB-resistant E. coli transformed with this plasmid were sensitive to chloramphenicol (Cam^S). A BP-recombination reaction was set up between the amplified attR4-attR3 Gateway cassette and pDONR221_Cam^S to create pDONR221-R4-R3 (see details under methods). The att sites involved in the reaction are marked with blue squares. Selection with LB medium containing kanamycin and chloramphenicol (Kan/Cam) yielded colonies containing the recombined plasmid. B. Adaptation of destination expression vector pEF5/FRT/V5-DEST for MultiSite Gateway recombination reactions. The same inactivating mutation in the CAT gene as above (red X) was introduced into the commercially available single-site destination expression vector pEF5/FRT/V5-DEST to generate a Cam-sensitive version (pEF5/FRT/DEST-Cam^S). The mutant plasmid was subjected to an LR-recombination reaction with pDONR221-R4-R3 in order to replace its original Gateway cassette with the attR4-attR3 MultiSite recombination cassette in pDONR221-R4-R3 (endowed with a wild type CAT gene). The att sites involved in the reaction are marked with green squares. Selection of the colonies containing recombined pEF5/FRT/V5-DEST (pEF5/FRT/-DEST-R4-R3) was carried out with LB medium containing ampicillin and chloramphenicol (Amp/Cam). Only the features in the pEF5/FRT/V5-DEST plasmid map that are relevant for the reaction are shown.