SummaryComprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino‐terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl‐terminal tag. We developed a streamlined protocol for high‐throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway™ recombinational cloning system. The protocol is straightforward to set up in large‐scale ORF cloning projects, and is cost‐effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.