A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes

A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes. This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101. The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker. To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts. The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes. Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts. An M13 ori allows the production of single-stranded DNA.