A cassette with seven unique restriction sites, including octanucleotide sequences: extension of multiple-cloning-site plasmids.

A 27-bp synthetic DNA cassette was constructed which contains the restriction sites of the two rare-cutter enzymes NotI and SfiI and, in an overlapping arrangement, those of five enzymes with 6-bp recognition sequences: ApaI, BalI, NdeI, SacII, XmaIII. The protruding termini of the fragment allow its insertion into any EcoRI-cut DNA creating a new EcoRI site at one side of the cassette only. This fragment was integrated into the pUC18-like multiple-cloning-site (MCS) plasmids pTZ18R and pTZ19R, producing a set of vectors which carry seven additional unique restriction sites (giving a total of 17) within their MCS. They still provide the capabilities of simple recombinant selection by blue/white coloured colonies, creation of single-stranded DNA in the presence of a helper phage, and in vitro transcription of cloned DNA using T7 RNA-polymerase. Plasmids with two copies of the DNA cassette inserted into their MCS were also constructed. Beside the advantages they provide in some cloning procedures, these latter plasmids, which carry a tandem repeat, are valuable sources of related 27-bp fragments, with features similar to the original but with different cloning termini.