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Gateway vectors have been extensively developed to facilitate gene cloning in numerous species; however, a universal system that is compatible for multiple organisms was lacking. As a multipurpose expression vector, pCS2+ backbone-based expression plasmids are widely used for high-level expression of messenger RNAs (mRNAs) or proteins in mammalian/avian culture cells or Xenopus/zebrafish embryos. To date, a suite of vectors with pCS2+ backbone applicable for Gateway cloning system were unavailable yet. Here, we generated a set of Gateway destination vectors, named as pGCS (plasmids of Gateway in pCS2+) vectors, which can be fused to a choice of frequently used amino- or carboxyl-terminal tags, including MYC, HA, FLAG, His, GST, as well as eGFP fluorescent epitope. The systematic generation of this set of pCS2+ backbone-based Gateway destination vectors allows for in vitro recombination of DNA with high speed, accuracy, and reliability compared with the traditional 'digestion-ligation' cloning approach. Thus, our system accelerates the production of functional proteins, which could be widely expressed in a large variety of vertebrate organisms without tediously transferring genes into different expression vectors. Moreover, we make this series of Gateway vectors available to the research community via the non-profit Addgene Plasmid Repository.

Construction of a series of pCS2+ backbone-based Gateway vectors for overexpressing various tagged proteins in vertebrates