Generating an Entry clone using a Gateway BP reaction. (A) A Gateway BP reaction uses BP clonase enzymes to recombine an attP site with an attB site to generate an attL site and an attR site, whereas LR clonase performs the reverse recombination. (B) attP, attL, and attR sites have recognition “arms” on either/both sides of the “recognition region” (box with arrowhead), whereas attB sites have no arms. These arms contain binding sites for the recombination enzymes and are named “left” and “right” with respect to the asymmetric overlap: attL sites have an arm 5′ (or left) of the overlap (white box), attR sites have an arm 3′ (or right, blue box), and attP sites have both arms. (C) attB sites are ~25 bp and feature a central 7-bp “asymmetric overlap” (boxed) that determines where DNA is cut and rejoined. attB0 is the naturally occurring site in the E. coli genome that is used by bacteriophage λ (Hartley et al. 2000). Mutagenesis has been used to generate the other varieties of attB sites.