MegaGate: A toxin-less gateway molecular cloning tool.

Gateway cloning employs the use of the ccdb toxin and has low colony numbers, making it difficult to apply at scale to clone libraries of cDNA vectors. In this protocol, we describe MegaGate, a toxin-less Gateway technology capable of robust cDNA library cloning that is efficient, cheap, and scalable. MegaGate eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning and is 99.8% efficient with high colony numbers. For complete details on the use and execution of this protocol, please refer to Kramme et al. (2021).