Plasmids for recombination-based screening

Gene. 1991 Sep 30;106(1):97-101. doi: 10.1016/0378-1119(91)90571-r.

Abstract

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage lambda / genetics
  • Base Sequence
  • Chloramphenicol Resistance / genetics
  • DNA / genetics
  • DNA, Viral / genetics
  • Genes, Viral
  • Genetic Markers
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides
  • Plasmids*
  • Recombination, Genetic*
  • Replicon

Substances

  • DNA, Viral
  • Genetic Markers
  • Oligodeoxyribonucleotides
  • DNA