Patent ID: 6924146

Claim:
An insertional mutagenesis system useful in constructing vectors for targeted mutagenesis of mammalian cells by homologous recombination, said insertional mutagenesis system comprising: (a) a linear lambda vector for cloning of genomic DNA into a site flanked on each side by negative selection markers, wherein a negative selection marker is a gene which causes cytotoxicity when stably expressed and active in a cell; and (b) a vector for insertion of a positive selection cassette into cloned genomic DNA, wherein a positive selection cassette encodes a gene which when stably expressed confers resistance against certain pharmacological toxins, said vector comprising: (1) an E. coli origin of replication; (2) an antibiotic resistance gene; (3) a selectable marker suitable for use in yeast; (4) a positive selectable marker, wherein a promoter element is operatively positioned 5′ to said positive selectable marker and a polyadenylation site operatively positioned 3′ to said positive selectable marker; (5) distinct Sfil restriction endonuclease sites flanking each side of the positive selectable marker so that said marker can be exchanged for another positive selection marker by directional cloning, wherein said SfiI sites are distinct in having different internal sequences which yield distinctive restriction fragment ends following Sfil digestion; (6) distinct restriction endonuclease sites for the excision of the positive selective cassette prior to ligation of synthetic deoxynucleotides to the cassette ends; and (7) restriction endonuclease sequences flanking the bacterial and yeast sequences to facilitate the removal of these sequences from the vector.