Restriction digests of the clone give the following sizes (kb): EcoRI--4.6, 2.0; HindIII--4.6, 1.0, 0.9; PstI--6.4. This cassette is inserted into the HindIII site (original nt 2272) of the ATS transposase-containing EcoRI fragment of pNK2881, ATCC 77351. Construct useful for general transposon mutagenesis in Escherichia coli. It also permits generation of promoter fusions of target genes to Plac-UV5, if the transposon inserts in the correct orientation. pBR322 was modified by deletion of bp 75-2352 and destruction (by filling in) of the HindIII site. Contains the ats1 ats2 transposase gene that permits relaxed insertion specificity (altered target specificity, ATS). Expression is regulated by the Ptac promoter, inducible by IPTG. The transposase segment extends from IS10R to the EcoRI site at nt 3140 of Tn10, with a deletion of nucleotides 1329-1942 to remove the transposase binding site. Contains a 1.9 kb mini-Tn10 cassette conferring kanamycin resistance, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites. Differs from pNK2859 (ATCC 77338) by also carrying a Plac-UV5 promoter in a BamHI fragment downstream of kanR and oriented so that transcription is in the same direction as the kan promoter out across the transposon end. 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 - ATS transposase - mini-Tn10 kan Plac-UV5 - EcoRI - pMB1 ori - ampR. |
