Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors.
Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (
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240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101
repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for
DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic
assay to suppress temperature-sensitive mutants of
ffh, encoding the protein component of the
Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.