Restriction digests of the clone give the following sizes (kb): EcoRI--2.15, 2.0, 1.1; HindIII--5.2; PstI--5.2; BamHI--5.0, 0.26. Construct useful for general transposon mutagenesis in Escherichia coli. pBR322 was modified by deletion of bp 75-2352 and destruction (by filling in) of the HindIII site. Contains the wild type transposase gene regulated by the Ptac promoter, inducible by IPTG. The transposase segment extends to the EcoRI site at nt 3140 of Tn10. Contains a 0.4 kb mini-Tn10 cassette conferring supF activity, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites. The 248 bp supF fragment can be excised by BamHI. Transcription from its promoter is opposite to that from Ptac. The mini-Tn10 cassette is inserted into the HindIII site (original nt 2272) of the wild-type transposase-containing EcoRI fragment. Transcription from the Ptac promoter is in a direction opposite to that of amp. The order of the major features in this plasmid is: EcoRI - Ptac - Tn10 transposase - supF - EcoRI - pMB1 ori - ampR. |
