Abstract: There is provided a non-PCR based method for construction of a DNA recombination fragment with necessary flanking regions homologous to a desired site of genetic manipulation within a target DNA required for recombination-based manipulation of said target DNA, comprising the steps of: A) identifying a desired site of insertion for a genetic element in said target DNA; B) locating an endogenous, native, half-site of a selected restriction endonuclease present upstream of the site within the DNA to be targeted for genetic manipulation and thereby defining the 5? extent of an upstream homology region; C) locating an endogenous, native, half-site of the same selected restriction endonuclease present downstream of the site within the DNA to be targeted for genetic manipulation and thereby defining the 3? extent of a downstream homology region; D) synthesising a flanking region cassette resulting in juxtapositioning of the upstream and downstream half-sites thereby causing formation of complete restriction site for