Lentiviral expression clones, which contain long direct repeats, often show dramatic instability in Escherichia coli, leading to difficulties in obtaining valid clones. We show that the reduced-genome E. coli strain MDS42 is capable of stabilizing lentiviral expression clones containing direct repeats, and outperforms many commonly used cloning strains for this purpose. In addition, the strain has several characteristics that make it highly amenable for use in recombinational cloning systems.