This cassette is inserted into the HindIII site (original nt 2272) of the ATS transposase-containing EcoRI fragment of pNK2881, ATCC 77351. The mini-Tn10 tetR cassette is unstable because a portion of the tetR marker inside the transposon is directly repeated in adjacent sequences outside the transposon. The plasmid should be propagaged in a recA- host, and the presence of the 2.9 kb BamHI fragment containing the cassette should be verified before use. The tetR marker SHOULD NOT be used for selection, as it is not effective in multiple copies. 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 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 2.9 kb mini-Tn10 cassette conferring tetracycline resistance, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites. 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 tetA tetR - EcoRI - pMB1 ori - ampR. |
