Cloning and Modification of Tn4001 Transposon

Tn4001mod on plasmid pISM2062 [28 (link)] was modified by adding a 6xHis-tag and a multiple cloning site: a 51-bp DNA fragment, created by annealing oligonucleotides HisC-f and HisC-r, was inserted between the BamHI and SmaI cleavage sites of pISM2062, resulting in plasmid pTnHis. The M. gallisepticum gene mgc2 was then amplified by PCR using genomic DNA of strain Rlow as template and primers ISM-mgcF and ISM-mgcR, and subcloned into pTnHis using the BamHI and SphI cleavage sites. The resulting plasmid, pTHmgc, was linearized with NotI, treated with the Klenow fragment of DNA polymerase I (New England Biolabs GmbH, Frankfurt/Main, Germany) to fill in the 5′ overhang, and subsequently digested with BamHI. A 1093-bp fragment was gel-purified and ligated to a 3.5-kb fragment of plasmid pINT [27 (link)], obtained after digestion with BamHI and SfoI.