Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis.

•The efficiency of using Golden Gate method to build knockout vector is more than 85%.•The expression of I-SceI endonuclease promoted homologous recombination.•The allelic-exchange schemes driven by I-SceI nuclease at rates of 85%–100%.•The Golden Gate method and the counter selectable marker were combined innovatively.