The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described. They all contain the pACYC184 origin of replication enabling them to replicate in E. coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E. coli and mycobacteria. They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E. coli. A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis.