Generating an Entry clone using a Gateway BP reaction. The creation of Entry clones, shown here, involves propagating a Donor vector in DB3.1 bacteria, amplifying a polymerase chain reaction (PCR) product with compatible attB sites, and then setting up a Gateway BP reaction. The BP clonase enzymes recombine the attB and attP sites, replacing the Gateway cassette with the amplified insert, which is now flanked by attL or attR sites depending on the configuration of the DNA fragments and vectors. The reaction shown on the left generates a promoter Entry clone, whereas that shown on the right generates an ORF Entry clone. Note that unidirectional cloning is ensured because attB1 sites only recombine with attP1 sites, attB2 sites with attP2 sites, and so on. After the BP reaction mixture is transformed into a standard bacterial cloning strain (such as DH5α), only the Entry clone will confer growth. Note that, typically, the asymmetric overlap is oriented 5′–3′ toward the insert, but att sites can also function in the reverse orientation and are then given the designation “R” (e.g., attB1R and attP1R). These reverse att sites are important for generating Entry clones that can be used in multisite LR reactions (Fig. 4).