The invention provides a method for altering the sequence of a target nucleic acid molecule, said method comprising the steps of: a) introducing a nucleic acid fragment into the target nucleic acid molecule by homologous recombination, wherein the nucleic acid fragment comprises: i) a first region of homology to the target nucleic acid molecule; ii) a first recognition sequence for a Type IIS nuclease; iii) a selectable marker; iv) a second recognition sequence for a Type IIS nuclease; v) a second region of homology to the target nucleic acid molecule; wherein components i) to v) are ordered from 5' to 3'; and a replacement nucleic acid sequence positioned between the first and second regions of homology but flanking the recognition sequences for the Type IIS nucleases; b) selecting for the incorporation of the replacement nucleic acid using the selectable marker, and c) cleaving the product of step b) with Type IIS nucleases such that the selectable marker is excised, to produce a linear target fragment including the desired replacement sequence.