Abstract: The present invention relates to a homing endonuclease which originates from Chlamydomonas eugametos, and was overproduced in E. coli, purified and characterized. The homing endonuclease of the present invention recognizes and cleaves degenerate double-stranded DNA at a specific recognition site; it particularly recognizes and cleaves 15, 17 and 19 nucleotide sequences. The cleavage of target DNA by this endonuclease produces a 4 nucleotide extension with a 3' OH overhang. A method to use the endonuclease of the present invention to cleave DNA fragments useful for gene mapping is also disclosed.