a). The structure of the FRT site. The FRT site consists of three 13 bp symmetry elements (horizontal arrows, a, b, and c, red letters) to which Flp binds in a site-specific manner. The 'a' and 'b' elements are in inverted orientation while the 'b' and 'c' elements are in direct orientation. The 'c' element is dispensable for recombination function but was included in all the substrates used here. The 'a' and 'b' elements are separated by an 8 bp asymmetric core region (open box, blue letters) across which recombination takes place. The vertical arrows indicate the two cleavage sites . Flp makes a pair of staggered breaks at these sites, covalently attaching itself to the 3'-phosphoryl C residue at each site via the catalytic tryosine, residue no. 343. b). General strategy for Flp double cross cloning. The receptor plasmid has the lacZ cassette flanked by inverted FRT sites. The gene of interest is PCR-amplified with primers that also contain inverted FRT sites at their ends. Flp promotes a double crossover and replaces the lacZ cassette with the gene of interest. The receptor plasmid (left) causes its host bacterium to make a blue colony when plated on the indicator dye X-Gal. The desired plasmid (right) causes a white colony. Because the FRT sites are in inverse orientation, the gene of interest is not cut out. Rather Flp inverts its orientation so that both orientations are usually obtained.