FIG. 2.

Design of template plasmids for various reporter fusions. Core vector p2795 contains the aph resistance cassette flanked by FRT sites. A multiple cloning site (MCS) with a large number of unique sites allows integration of reporter genes. A collection of template vectors with various reporter genes was constructed (plasmids p3121 to p3176). Linear targeting DNA was generated by PCR with primer pairs consisting of forward primers (For) complementary to the reporter and common reverse (Rev) primers. The design of primer pairs for the amplification of targeting DNA is shown. Examples for the generation of translational fusions are shown as in Fig. 1B. Primers typically contain 40-nucleotide (nt) sequences complementary to the target region. The following 20 to 22 nt of the forward primers are specific to one of the reporter genes; the 40-nt sequence was in frame with the ORF of the reporter gene. The reverse primers include a 21-nt sequence complementary to a common priming site of the template plasmids that could be used with each reporter in the series.