title: Plasmids for recombination-based screening.
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.
authors.affiliation: Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650.
authors.forename: G D
authors.initials: GD
authors.lastname: Stewart
authors.forename: M A
authors.initials: MA
authors.lastname: Hauser
authors.forename: H
authors.initials: H
authors.lastname: Kang
authors.forename: D P
authors.initials: DP
authors.lastname: McCann
authors.forename: M M
authors.initials: MM
authors.lastname: Osemlak
authors.forename: D M
authors.initials: DM
authors.lastname: Kurnit
authors.forename: A J
authors.initials: AJ
authors.lastname: Hanzlik
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002702:Chloramphenicol Resistance / Q000235:genetics; D004247:DNA / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005819:Genetic Markers; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011995:Recombination, Genetic; D012093:Replicon
chemical_list: D004279:DNA, Viral; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D004247:DNA