RRC ID 30051
Author Vieira J, Messing J.
Title The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.
Journal Gene
Abstract A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.
Volume 19(3)
Pages 259-68
Published 1982-10-1
DOI 10.1016/0378-1119(82)90015-4
PII 0378-1119(82)90015-4
PMID 6295879
MeSH Base Sequence Cloning, Molecular* DNA Restriction Enzymes DNA Transposable Elements DNA, Recombinant / metabolism* Drug Resistance, Microbial Escherichia coli / genetics* Kanamycin / pharmacology Mutation* Plasmids*
IF 2.638
Times Cited 5617
Prokaryotes E. coli ME8643