The present invention includes a method of generating a mutant Herpes Simplex Virus (HSV). In one embodiment, the generated HSV genome includes nucleic acid encoding a nucleic acid sequence of interest. In one step, a nucleic acid vector is provided which includes a nucleic acid encoding first and second site specific recombination sequences and a nucleic acid encoding a nucleic acid sequence of interest between said site specific recombination sequences an HSV is provided, the genome of which comprises third and fourth site specific recombination sequences In another step, the nucleic acid vector and HSV are contacted together with one or more recombinase enzymes capable of catalyzing site specific recombination between the site specific recombination sequences of said nucleic acid vector and said HSV. Another step includes identifying HSV containing the nucleic acid sequence of interest. In some embodiments, the methods are conducted in a cell-free system.