(A) The cloning vector pYAC3. (B) To clone with pYAC3, the circular vector is digested with BamHI and SnaBI. BamHI restriction removes the stuffer fragment held between the two telomeres in the circular molecule. SnaBI cuts within the SUP4 gene and provides the site into which new DNA will be inserted. Ligation of the two vector arms with new DNA produces the structure shown at the bottom. This structure carries functional copies of the TRP1 and URA3 selectable markers. The host strain has inactivated copies of these genes, which means that it requires tryptophan and uracil as nutrients. After transformation, cells are plated onto a minimal medium, lacking tryptophan and uracil. Only cells that contain the vector, and so can synthesize tryptophan and uracil, are able to survive on this medium and produce colonies. Note that if a vector comprises two right arms, or two left arms, then it will not give rise to colonies because the transformed cells will still require one of the nutrients. The presence of insert DNA in the cloned vector molecules is checked by testing for inactivation of SUP4. This is done by a color test: on the appropriate medium, colonies containing recombinant vectors (i.e. with an insert) are white; non-recombinants (vector but no insert) are red.