A NEW DNA CLONING SEQUENCING VECTOR WITH A BUILT-IN MECHANISM FOR GENERATION OF NESTED DELETIONS USING TRANSPOSON TN3/

We have constructed a new cloning/sequencing vector suitable for seque ncing of DNA fragments too long to be sequenced in a single step. This vector plasmid contains inverted repeats (IR) of transposon Tn3, the kil and c1857 genes of phage lambda, and multiple cloning sites (MCS). Escherichia coli cells harboring plasmids containing nested deletions from one of the Tn3 IR ends, across kil, to variable end points, can be positively selected by plating at 42 degrees C. The deletion produc ts, fractionated according to their size by agarose-gel electrophoresi s, can be sequenced by using a synthetic primer whose 3'-end is locate d within the Tn3 IR, and the total sequence of the insert can be const ructed from the partial sequences.