This protocol describes using the Gateway recombinatorial cloning system to simultaneously transfer a promoter and an open reading frame (ORF) from two different Entry clones into the same Destination vector using LR enzymes. A multisite cloning reaction transfers the inserts from multiple Entry clones into a single Destination vector. This type of recombination is much less efficient than transferring a single DNA fragment; however, the variety of Destination clones that can be generated in this manner is vast. In this example protocol, we describe using pDEST-MB14 to make a Destination clone that features a promoter fragment fused upstream to an ORF that is cloned in-frame with a carboxy-terminal green fluorescent protein (GFP) moiety encoded by the plasmid backbone. This method can be used as a guide for other multisite cloning reactions.