Expression-ready cDNA clones, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of the gene products. To create a resource of human gene products, we attempted to systematically convert original cDNA clones to expression-ready forms for recombinant proteins. For this purpose, we adopted a rare-cutting restriction enzyme-based system, the Flexi cloning system, to construct ORF clones. Taking advantage of the fully sequenced cDNA clones we accumulated to date, a number of sets of Flexi ORF clones in a 96-well format have been prepared. In this section, two methods for the preparation of Flexi ORF clones in a 96-well format are described. A protocol for transferring ORFs between Flexi vectors in a 96-well format is also described. We believe that the resultant clone set could be successfully used as a versatile reagent for functional characterization of human proteins.