Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling (AfCS), we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomic applications. To date, we have generated over 3,000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search and blast AfCS plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here, we describe the cloning, databasing and application of this proteomic resource for large-scale subcellular localization screens in mammalian cell lines.