Construction of reporter gene fusions through a three-molecule LR reaction. The Expression Clone, containing the promoter::gfp reporter gene fusion, is generated through a series of three steps (bold arrows) that involve specific recombination reactions (crosses). In Step 1, recombination sites of the appropriate specificity (determined by the sequence of the core 7 nucleotides within the att sites) are appended to the desired DNA segment by PCR. In Step 2, the Promoter Entry Clone is generated by BP recombination of attB4-promoter-attB1 PCR product with the attP4 and attP1R sites, respectively on pDONR P4P1R. The GFP Entry Clone is generated in a standard Gateway reaction by BP recombination of an attB1-ORF-attB2 PCR product with the attP1 and attP2 sites, respectively, on pDONR201. Selection for kanamycin resistance and against ccdB, upon DH5α transformation with the BP reactions, ensures the correct Entry Clone plasmids are obtained. In Step 3, these Entry Clones and the desired Destination Vector (pDEST R4R2) are combined in a three-molecule LR recombination. In this reaction, which depends on the specificity of the attL1 with attR1, attL2 with attR2, and attL4 with attR4 recombinations, the promoter and GFP DNAs are linked and transferred into the Destination Vector, pDEST R4R2. Selection for ampicillin resistance and against ccdB, upon transformation of DH5α, ensures only the desired Expression Clone is obtained.