For cloning, the vector is restricted with a combination of BarnHI and SnaBI. BamBI cleaves the vector at the junctions of the two TEL sequences with the fragment that is used to circularize the vector for propagation in E. coli; this fragment is discarded.

The enzyme SnaBI recognizes the single sequence 5'T ACGT A3' located in SUP4 and produces blunt-ended cleavage, thereby generating two arms of the YAC, each ending in a TEL sequence. The DNA insert, therefore, must have blunt ends; it is integrated within SUP4 to generate the linear YAC.

The recombinant YAC is introduced into TRP 1^- URA3^- yeast cells by protoplast transformation; transformed cells are selected by plating them onto the minimal medium: only those cells are able to grow on this medium that have correctly constructed YAC containing one left and one right arm of each chromosome.

The yeast genes present indifferent yeast vectors can become integrated into the host genome; this is called permanent transformation. It generally occurs through homologous recombination between the gene present in a vector (e.g., LEU2) and that present in the yeast chromosomes (e.g., LEU2-). Rarely, the gene may become inserted at a random chromosome site.

The homologous recombination may occur by regular crossing over or it may involve gene conversion (a non­reciprocal recombination). Vectors have been devised for high frequency stable transformation; such vectors are introduced in yeast cells in linear form and contain at their both ends sequences that are homologous to those found at the target site (where the gene present in the vector is to be integrated) in the yeast genome.

Such vectors permit integration of any specified DNA sequence at the desired site in yeast genome, i.e., they allow site-specific transformation (= integration) of genes.