Figure 4. Multiple segment assembly using Gateway.

Multisegment Gateway cloning works by the same means as single segment Gateway (see Figure 2) except that it requires additional sets of att sites of slightly different sequence. The att sites flanking each segment determine the order of the segments in the final recombined vector. In round 1, Entry clones are generated by flanking PCR products with unique attB sites and recombining them with vectors containing unique attP sites. The resulting entry clones can then be used to generate multisegment inserts. In round 2, Entry clones containing the different DNA segments flanked by attL and attR sites are recombined with an attR containing destination vector. The destination vector (Dest) also contains the ccdB positive selection marker (Bernard et al., 1994). After a successful LR recombination, the ccdB cassette is replaced by the full insert, which contains all three segments separated by attB scar sites (Figure 5D).