Plasmids for recombination-based screening.
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.
Stewart
G D
GD
Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650.
Hauser
M A
MA
Kang
H
H
McCann
D P
DP
Osemlak
M M
MM
Kurnit
D M
DM
Hanzlik
A J
AJ
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
D004279:DNA, Viral; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D004247:DNA