A plasmid, pJS133, was constructed which contains a promoterless cat gene encoding chloramphenicol (Cm) acetyltransferase (CAT) conferring Cm resistance. This improved cat cassette, derived from the cat gene in plasmid pKK232-8 [Brosius, Gene 27 (1984) 151-160], offers several advantages over currently available cat cassettes. Expression of cat from this cassette reflects only transcriptional activity because of translational stops in all three reading frames, and possesses facile insertion/excision properties employing the flanking polylinker restriction sites. In plasmid pJS133, this cassette can be obtained by a single digestion with SmaI, HindIII, SalI, BamHI or PstI restriction enzymes. Additionally, 28 different cat cassettes with unique flanking restriction sites can be created by the appropriate double digestions.