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In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a Δasd::aph allele. Attempts to replace the wild-type asd gene with the Δasd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains β-galactosidase expressed from a mycobacteriophage promoter and Δasd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the β-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10-5 per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.