An international team of researchers has embarked on the creation of a synthetic eukaryotic genome and now report the synthesis of a redesigned Saccharomyces cerevisiae chromosome. Boeke and colleagues built a fully functional chromosome III (which they term synIII) that contained hundreds of alterations, including the removal of introns, transposons and tRNA genes to increase the stability of the re-engineered chromosome. In addition, 98 loxP sites were inserted to enable genetic reshuffling of the chromosome as desired. Replacement of chromosome III with synIII generated a viable yeast strain that had negligible fitness costs and a highly similar transcriptome compared with the original strain. The synthesis of complete bacterial and viral genomes has already been accomplished, but this is a landmark achievement that paves the way for the construction of entirely synthetic, tailor-made eukaryotic genomes.