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Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae
Author(s) -
Goldstein Alan L.,
Pan Xuewen,
McCusker John H.
Publication year - 1999
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199904)15:6<507::aid-yea369>3.0.co;2-p
Subject(s) - ura3 , biology , plasmid , heterologous , gene , saccharomyces cerevisiae , selectable marker , genetics , candida albicans , kanamycin
Heterologous gene replacement cassettes are powerful tools for dissecting gene function in Saccharomyces cerevisiae. Their primary advantages over homologous gene replacement cassettes include reduced gene conversion (leading to efficient site‐specific integration of the cassette) and greater independence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene ( kan r ), which confers resistance to G418 (Wach et al ., 1994). One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains with more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes by replacing the kan r ORF from plasmids pFA6–kanMX3 and pFA6–kanMX4 with the Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 (CaURA3MX3) are identical to the kanMX cassettes in all other respects but have the added advantage of being counterselectable and therefore readily recyclable in S. cerevisiae . Copyright © 1999 John Wiley & Sons, Ltd.