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. 2006 Jan;72(1):291–297. doi: 10.1128/AEM.72.1.291-297.2006

FIG. 2.

FIG. 2.

Construction of plasmids used for tnpA promoter analysis. (A) Nucleotide sequence upstream of the tnpA gene of IS1071. Capital letters indicate the 110-bp IR sequence of IS1071. The boxed region (145 bp) was amplified by PCR with primers 1071pro1 and 1071pro3 and used to construct pMS0321. (B) Construction of plasmids. Abbreviations for restriction endonucleases are as follows: B, BamHI; Bg, BglII; P, PstI; S, SalI. Plasmid pCB182 is a promoter-probe vector and carries a multiple-cloning site (MCS) between the galK and lacZ genes, each of which lacks its promoter sequence (22). The BglII-PstI fragment of pCB182 that carried the promoterless lacZ gene was cloned between the BamHI and PstI sites in pBBR1MCS (16) to generate pMS0326. The 145-bp sequence of IS1071 (white arrowhead) flanked by SalI and BglII sites (see panel A) was inserted between the corresponding sites in pCB182 to generate pMS0321. Subsequent cloning of the BamHI-PstI fragment of pMS0321 containing the lacZ gene between the corresponding sites in pBBR1MCS led to the construction of pMS0324.