Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning.
The Gateway technology is becoming an increasingly popular method for cloning ORFs by recombination. It allows the transfer of any ORF flanked by specific recombination sites into any vectors harbouring the corresponding sites. Here we describe the construction of a set of 20 Saccharomyces cerevisiae Gateway compatible vectors. These plasmids bear an URA3 or TRP1 selection marker. They are designed for expression without tag sequence or for C- or N-terminal protein tagging with 3HA (haemagglutinin), 13MYC, 4TAP (tandem affinity purification) or GST (glutathione S-transferase) epitopes. The centromeric vectors allow expression of DNA sequence in yeast under tetracycline-regulatable promoters, while expression from the high copy vectors is driven by PGK promoter. To test their applicability, the genes encoding the RNA polymerase I subunit Rpa12p or the TFIIS transcription factor were cloned in these vectors. Their expression was demonstrated using Western blotting or complementation assays.
Van Mullem
Vincent
V
Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. vincent.vanmullem@fundp.ac.be
Wery
Maxime
M
De Bolle
Xavier
X
Vandenhaute
Jean
J
D003001:Cloning, Molecular / Q000379:methods*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D035165:Transcription Factors, General / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D004271:DNA, Fungal; D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; C423311:RPA12 protein, S cerevisiae; D012318:RNA Polymerase I