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Mycobacterium paratuberculosis promoter-containing clones were isolated from a genomic DNA library constructed in the transcriptional-translational fusion vector pYUB76. The promoter-containing DNA fragments were identified in the surrogate host Mycobacterium smegmatis by expression of the promoterless lacZ reporter gene of pYUB76. The expression signals exhibited a wide range of strengths, as indicated by their corresponding β-galactosidase activities. Eight clones were sequenced and characterized further. Predicted open reading frames and codon usage were identified by computer analysis. Database searching for related sequences using the BLAST method revealed no homologies. Transcriptional activity was measured by slot-blot hybridization with steady-state RNA isolated from lacZU M. smegmatis clones. Primer extension analysis identified the transcription start sites within the cloned fragments. The promoter regions characterized in this study were used to establish a consensus promoter sequence for M. paratuberculosis. M. paratuberculosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centred approximately 35 and 10 bp upstream from the transcription startpoints do not correspond to the consensus hexanucleotides of Escherichia coli promoters.