Restriction digests of the clone give the following sizes (kb): BamHI--24.0, 14.0, 9.6; EcoRI--19.0 (doublet), 9.5; HindIII--23.0, 9.0, 5.6 (doublet), 4.5; SalI--20.0, 9.8, 8.8, 5.6, 5.0. Excision of plasmids requires hosts expressing the tnpR resolvase, that recognizes the 130 bp res resolution sites in the vector. Escherichia coli HB101 F'lac::Tn1739tnpR (ATCC 47068) expresses high levels of tnpR and encodes lambda cI to repress expression from lambda vector fragments (and possible cell lysis) after excision of the plasmid. Plasmids can be excised by infecting ATCC 47068 with the recombinant lambda phage, inducing tnpR expression using IPTG, and plating on media containing kanamycin. Lambda replacement vector useful for genomic library construction. Permits Spi+ selection and excision of cloned fragments as autonomously replicating plasmids. The excised plasmid (pEX) contains the Rts1 origin of replication, a kanamycin resistance gene, the cloned insert (or vector replacement fragment), and a single res site. |
