Robust multi-type plasmid modifications based on isothermal in vitro recombination

A robust strategy for multi-type plasmid modifications is developed based on the isothermal in vitro recombination technology, by which any combination of the sequence modifications can be efficiently achieved in plasmids at any desired position in a seamless manner. As an example, we showed that a plasmid modification with insertion of a GFP gene, deletion of a 623-bp fragment, and substitution of an ampicillin resistance gene by a kanamycin resistance gene was accomplished simultaneously by this method. Therefore, the isothermal in vitro recombination-based multi-type plasmid modification strategy is a useful approach for broad application prospects in molecular biology studies. •A simple strategy for complex modifications of plasmids is developed.•The method is based on the isothermal in vitro recombination technology.•Multiple types of sequence modification of plasmid can be achieved simultaneously.•It is restriction endonuclease-free, seamless and almost zero-background.