Restriction digests of the clone give the following sizes (kb): EcoRI/BglII--3.7, 0.5; BamHI--4.2; XbaI--4.2; NotI--2.4, 1.8. Vector containing the attP sequence for integrating DNA into the lambda attachment site attB. Requires the lambda integrase (int) such as that provided by pLDR8 (ATCC 77357). When a recombinant construct is digested with NotI, the origin is separated from the insert. Each fragment has a distinct selectable marker. After religation to form origin-free circles, the products are transformed into bacteria with pLDR8. Expression of int in pLDR8 is regulated by lambda PR, by the temperature sensitive cI857 repressor. Replication of pLDR8 is also temperature sensitive. Therefore, at 30C replication of the plasmid is normal and int is not expressed. At 42C, int is expressed but pLDR8 is no longer replicated, resulting in loss of int after a few cell generations. The order of the major features in this plasmid is: NotI - ampR - ClaI/MCS/EcoRI - attP - NotI - pMB1 ori - kanR. |
