Abstract: The present invention relates to the easy cloning of multiple DNA fragments at multiple places in the vector by a single step recombination reaction. More specifically the present invention discloses the use of two recombination sites each having the same pair of recombination sequences and the recombination sites are placed in opposite direction, in order to prevent the recombination between the sites themselves, and offering the opportunity to clone different or the same DNA fragments in multiple sites of the vector. This method is very useful for high throughput cloning of for example a co-suppression vector, or a gene combination vector, or a promoter combination vector or a promoter-gene combination vector, or a gene silencing vector, or a polycistronic RNA vector, or a gene stacking vector, or a biderectional promoter, or combinatorial expression cassettes or a fusion protein.