Restriction digests of the clone give the following sizes (kb): HindIII--4.8; EcoRI--4.0, 0.9; BamHI--4.8. pMLP9 is the same as pMLP8, except that a BglII site has been added to pMLP9. 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. This is a postive selection cloning vector which will confer chloramphenicol resistance (recommended concentration is 10ug/ml) only when an insert has been successfully cloned into the cI repressor gene. Recommendation for verfication: HindIII; BglII; SmaI. 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. |
