Restriction digests of the clone give the following sizes (kb): EcoRI--3.4, 0.89; PstI--4.27; EcoRI/HindIII--2.45, 0.89, 0.75. The cml cassette can be isolated by digestion with one enzyme (BamHI), by double digestion yielding blunt ends (HincII/SmaI), or by double digestion yielding overhangs for directional cloning. pUC18CML (ATCC 37717) and pUC18CMR (ATCC 37718) differ only in the orientation of the cml cassette. In pUC18CMR, the lac promoter and the cml gene are in the same orientation. A plasmid containing the gene determining chloramphenicol resistance which is flanked by polylinker sites for ease in manipulation. cmlR sequences (pBR325 cut with BclI/blunt ended with Klenow/cut with HincII) were ligated to a 2.6 kb HincII fragment of pUC4K. The 1.6 kb BamHI fragment was cloned into M13mp18; a 1.6 kb KpnI/HindIII fragment was cloned into the BamHI site of pUC18. |
