Restriction digests of the clone give the following sizes (kb): KpnI--5.6, 2.0; SstI--7.6; BamHI--7.6. Replicates only in enterobacteria, and so functions as a suicide vector in other Gram negative hosts. A single BglII site within the gentamicin resistance gene (gtmR) allows insertion of other resistance genes (e.g. as a BamHI cassette). When induced by media containing 5% sucrose, sacB is lethal in a wide range of Gram negative bacteria, and thus permits selection for loss of the vector. IMPORTANT: To prevent amplification of a rearranged and/or deleted cosmid, we recommend streaking on LB + amp plates at 30C and picking small colonies for liquid culture. Suicide vector permitting gene replacement and mobilization into a wide range of Gram negative bacteria. Allows positive selection for integration. The order of the major features of the plasmid is: gtmR - KpnI/MCS/SstI - P15A - cos - traJ - oriT - sacB. |
