Method of cloning at least one nucleic acid molecule of interest using type IIS restriction endonucleases, and corresponding cloning vectors, kits and system using type IIS restriction endonucleases

摘要

The present invention refers to methodsof (sub)cloning at least one nucleic acid molecule of interest. One embodiment relates to a method of (sub)cloning at least one nucleic acid molecule of interest comprising a) providing at least one (replicable) Entry vector into which the at least one nucleic acid molecule of interest is to be inserted, wherein the at least one Entry vector carries two recognition sites for at least one first type IIS and/or type IIS like restriction endonuclease and wherein said at least one nucleic acid molecule of interest can be excised from the at least one Entry vector at two combinatorial sites with one (same) or more (different) cohesive ends that are formed by the at least one first type IIS or type IIS like restriction endonuclease, and b) providing an Acceptor vector, into which the at least one nucleic acid molecule of interest is transferred from the at least one Entry vector carrying the at least one nucleic acid molecule of interest, wherein said Acceptor vector comprises at least one recognition site for at least one second type IIS restriction endonuclease and/or at least one recognition sites for at least one type IIS like restriction endonuclease, and wherein said Acceptor vector provides two combinatorial sites identical to the two combinatorial sites present in the Entry vector. The inventions also relates respective cloning vector and kits.