Restriction digests of the clone give the following sizes (kb): EcoRI--6.0, 2.1, 1.5; HindIII--9.2, 0.82; PstI--10.0. Construct useful for generating operator fusions in which the promoterless lacZ gene is transcribed from the promoter of the gene into which it inserts. 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 4.8 kb mini-Tn10 cassette conferring kanamycin resistance and including a promoterless lacZ, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites. The mini-Tn10 cassette is inserted into the HindIII site (original nt 2272) of the wild-type transposase-containing EcoRI fragment. The order of markers is: EcoRI - Ptac - WT transposase - kanR - lacZ - EcoRI - ori - ampR. Transcription from the Ptac promoter is in a direction opposite to that of amp. |
