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Bacteria belonging to the Gram-positive actinomycete species, Rhodococcus erythropolis, are diverse not only in terms of metabolic potentials but the plasmids they encode. It was shown previously that the R. erythropolis AN12 genome harbors a 6.3 kb cryptic plasmid called pAN12, which is a member of the pIJ101 family of plasmids. Here we show that pAN12 is conjugatively mobilizable into other rhodococcal strains. A series of plasmid deletion constructs were tested for loss of mobility to identify the pAN12 cis-acting conjugation requirement. In this way, an approximately 700 bp region was found to be required for plasmid transmission. A small 61 bp element within this region confers mobility to an otherwise non-mobilizable plasmid. Unlike pIJ101, which encodes all necessary factors for transfer, pAN12 mobility is dependent on the presence of an AN12 megaplasmid, pREA400