Restriction digests of the clone give the following sizes (kb): EcoRI--26.0, 9.8, 3.0, 2.7, 1.8; HindIII-->23.0, 6.6, 3.9, 3.4, 1.6, 0.6; BglII--32.5, 7.6, 2.5, 0.7, 0.5; XbaI--24.5, 18.0. Construct useful for general transposon mutagenesis in Escherichia coli. The vector has mutations in replication genes, the repressor, and the integration system. Genotype: b522 cI857 Pam80 nin5. The EcoRI cloning site is between bp 21226 and 26104. 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 1.4 kb mini-Tn10 cassette conferring chloramphenicol resistance, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites. The order of the major features in this bacteriophage is: A-J - EcoRI - mini-Tn10 cam - ATS transposase - Ptac - EcoRI - b522 - cI857 - P80 - nin5. |
