Restriction digests of the clone give the following sizes (kb): EcoRI--2.8, 1.2; BamHI--2.75, 1.3; HindIII--2.7, 1.4. The nptII gene was modified by site-directed mutagenesis to remove PstI and SphI sites. An inverted repeat of the 271 bp NdeI/EcoRI fragment of pUC18 surrounds the kanR cassette. If this vector is ligated without a stuffer fragment, this inverted repeat prevents replication, thus providing positive selection for plasmids with inserts. Plasmid with an easily purified kanR cassette (flanked by inverted repeats of the pUC18 multiple cloning sequence) for construction. pBSL15 and pBSL86 differ in the orientation of the repeats. |
