Restriction digests of the clone give the following sizes (kb): HindIII--8.2; EcoRI--7.2, 0.96; BglII--8.2. The cytoplasmic chloramphenicol resistance protein does not negatively affect cell growth when overproduced (in contrast to cloning vectors that overexpress membrane associated tetR protein). The level of resistance to chloramphenicol differs substantially depending on the stage at which they are tested. The chloramphenicol resistance of the plasmid should be tested before cloning inserts. YE-type shuttle vector allowing positive selection of inserts by conferring chloramphenicol resistance (recommended concentration 10 ug/ml) when a sequence has been inserted into the cI repressor gene. A pair of phage fd rho-independent strong terminators downstream of the cmlR gene prevents transcription through the origin of replication. This stabilizes recombinant plasmid copy number when the PR promoter for the cmlR gene is derepressed. |
