A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli.
We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.
Schlieper
D
D
Institut für Genetik, Universität zu Köln, Germany.
von Wilcken-Bergmann
B
B
Schmidt
M
M
Sobek
H
H
Müller-Hill
B
B
D003001:Cloning, Molecular / Q000379:methods*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005804:Genes, Lethal; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial