Duplication of a region in the multiple cloning site of a plasmid vector to enhance cloning-mediated addition of restriction sites to a DNA fragment.

(occurring within SwaI) and BamHI restriction sites as well as compatible non-regenerating BamHI and EcoRI cohesive ends (Figure 1A). The oligonucleotides were ligated in the presence of T4 DNA ligase to the SuperCos 1 vector previously digested with BamHI and dephosphorylated. The DNA that had been subsequently digested with an excess of BamHI and fractionated on agarose gels was purified and then selfligated in the presence of T4 DNA ligase. Following transformation of E. coli XL1-Blue MR (Stratagene), plasmid DNA was extracted and analyzed for the presence of HindIII, PacI, SwaI, DraI and BamHI restriction sites. The characteristics of the resulting SuperCos/HPS vector are illustrated in Figure 1B. The vector was subsequently used for the construction of two cosmid libraries with BamHI or Sau3A partially digested BHV-1 DNA. Because of the presence of SwaI and PacI restriction sites closely flanking the BamHI cloning site, intact subgenomic inserts could be readily generated by a simple digestion of the cosmid recombinants (not shown). In addition, EcoRI and HindIII flanking sites could be used to identify the genomic regions that were included in the clones by comparing the restriction patterns obtained to the previously reported restriction maps (1). Because of its significantly improved MCS, the SuperCos/HPS vector will facilitate the characterization of large GC-rich DNA inserts. A similar strategy could be used to introduce GC-rich restriction sites for the analysis of ATrich DNA inserts.