Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments.

A plasmid cloning vector, pHSG664, has been constructed which is suitable for the direct-selection of transformed cells with recombinant DNAs. The plasmid contains the replicon and ApR-gene region of pUC9 ligated to the strA+ (rpsL+) gene derived from pNO1523 [Dean, Gene 15 (1981) 99-102]. The vector contains ten unique restriction sites: EcoRI, HpaI, PvuII, SphI, PstI, SmaI(XmaI), BamHI, SalI(AccI, HincII), XbaI and HindIII. Five sites (bold-face lettering) are located within the coding region of the strA+ gene. Any insertion at the five bold-faced sites or any nucleotide replacement involving the strA+ gene region and the other unique sites can be selected by Ap and Sm double selection in a strA- (SmR) strain such as E. coli HB101. Thus, this vector is useful for cDNA cloning at either the SphI or the PstI site with the G:C-tailing procedure, as well as for the cloning of double-digest DNA segments.