Abstract
We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 201-216 |
| Number of pages | 16 |
| Journal | Gene |
| Volume | 58 |
| Issue number | 2-3 |
| DOIs | |
| State | Published - Jan 1 1987 |
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All Science Journal Classification (ASJC) codes
- Genetics
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Plasmid construction by homologous recombination in yeast. / Ma, Hong; Kunes, Sam; Schatz, Peter J.; Botstein, David.
In: Gene, Vol. 58, No. 2-3, 01.01.1987, p. 201-216.Research output: Contribution to journal › Article
TY - JOUR
T1 - Plasmid construction by homologous recombination in yeast
AU - Ma, Hong
AU - Kunes, Sam
AU - Schatz, Peter J.
AU - Botstein, David
PY - 1987/1/1
Y1 - 1987/1/1
N2 - We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.
AB - We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.
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UR - http://www.scopus.com/inward/citedby.url?scp=0023615620&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(87)90376-3
DO - 10.1016/0378-1119(87)90376-3
M3 - Article
C2 - 2828185
AN - SCOPUS:0023615620
VL - 58
SP - 201
EP - 216
JO - Gene
JF - Gene
SN - 0378-1119
IS - 2-3
ER -