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Integrons are considered expression systems due to the presence of Pc promoters that drive gene cassette transcription. The role and configurations of Pc are well known in class 1 integrons; however, this region has not yet been identified in class 2 integrons. This study aimed to characterize the Pc promoter from class 2 integrons and to determine the effect of gene cassette position on transcription driven by this promoter.The class 2 cassette arrays from Klebsiella pneumoniae and Vibrio cholerae strains were determined by sequencing. Transcription analyses were performed by real-time RT–PCR and relative quantification was carried out by comparing the transcripts of each normalized gene inserted in the integron to each other. The resistance profile was determined by the disc diffusion method. The class 2 Pc promoter was characterized by 5′ rapid amplification of cDNA ends and promoter prediction programs.Sequence analysis revealed the presence of the dfrA1-sat2-aadA1-ybeA and sat2-aadA1-ybeA arrangements in K. pneumoniae and V. cholerae strains, respectively. Real-time RT–PCR showed that the transcription of the first cassettes was higher than that of distal ones in wild-type and recombinant strains. All strains were resistant, indicating cassette expression. The Pc promoter of class 2 integrons (−35 TTTAAT |16 bp| −10 TAAAAT) was determined based on in silico analyses and on the transcription start site sequence of the class 2 integron cassette array.The Pc from class 2 integrons was characterized for the first time and the cassette position effect on transcription was demonstrated.