An M13 vector library for cloning DNA with four nucleotide 3′ overhangs

Abstract

A flexible M13 vector library incorporating the BstXI site has been developed. DNA cut by any currently commercially available restriction endonuclease that generates a 4-nucleotide (nt) 3′ overhang can be ligated into a specific clone of the library. The BstXI enzyme recognizes a 6-bp bipartite palindromic sequence. The central nucleotides are not specified, and form a 4-base, 3′ overhang when cut by BstXI. 5′ CCANNNNN NTGG GGTN NNNNNACC 5′ Since the 4-base overhang formed is not part of the BstXI recognition sequence, it is possible to generate a library of 256 different clones by introducing the BstXI site. 151 of the possible 256-member library have been isolated, including all 13 M13BF clones in which the overhang formed by BstXI digestion is complementary to those formed by currently available restriction endonucleases. Of these 13 vectors, BstXI digestion of six clones results in nonpalindromic cohesive ends and should facilitate in vitro tandem gene amplification. The BstXI site is adjacent to the four codons corresponding to the factor Xa recognition sequence. Hence the vector library could facilitate the expression of a fusion protein that could be proteolytically cleaved by factor Xa.