Mixture of cell extract and method for site-directed cloning

The present invention provides novel reagents and a cloning procedure based on homologous recombination for the site-directed cloning of a DNA fragment to a vector at designed site(s). The cloning reagents are made of mixture of extracts from at least two different cell types, preferably a mixture made of extracts from wild-type E. coli and S. cerevisiae. Due to the activity of the mixture of cell extracts, recombination occurs between the 3′ and 5′-ends of the target DNA and at the ends of linearized vector, which facilitates in-frame construction of expression vectors.