Paste a list of target Gene names and/or Open Reading Frames (ORFs) separated by line breaks (<Enter>),or use the search field on the right to find and insert entries one-by-one.
The list can include a mixture of ORF and gene names. Duplicates will be removed from results.
Gene name is the standard name, e.g. TFC3. ORF is the systematic name, e.g. YAL001C.
Please notice that while the size of the gene list is not limited, inputs of large sets (>500 entries) will result in long processing time of up to a few minutes.
Knockout (KO) primers are intended to amplify a selection cassette from a plasmid, with flanking sequences directing it to recombine in place of the target gene.
Forward primer: 40 base pairs (bp) upstream to the gene’s start codon (ATG) (including the ATG in the primer), followed by the “forward primer” sequence of the transformation cassette (plasmid dependent).
Reverse primer: The reverse complement of 40bp downstream to the gene’s Stop-codon (including the Stop-codon in the primer), followed by the “reverse primer” sequence of the transformation cassette (plasmid dependent).
To verify correct insertion use WT Check primers, or 5UTR / 3UTR Check primers.
DAmP primers are intended to amplify a selection cassette from a plasmid, with flanking sequences directing it to recombine in place of the gene’s 3’UTR (immediately after the stop codon). The presence of the selection marker promoter which replaces the original gene’s 3’UTR perturbs gene expression, as described by Schuldiner et al. 2005 (References).
Note that it is recommended to perform the DAmP switch in diploid cells if the target gene is essential. Following sporulation the haploids have less suppressor mutations.
Forward primer: 40bp upstream to the gene’s Stop-codon (including the Stop-codon in the primer), followed by the “forward primer” sequence of the transformation cassette (plasmid dependent).
Reverse primer: The reverse complement of 40bp after the gene’s Stop-codon (excluding the Stop-codon from the primer), followed by the “reverse primer” sequence of the transformation cassette (plasmid dependent).
To verify correct insertion use C’-tag Check primers.
C’ tagging primers are intended to amplify a cassette containing a tag to be fused in frame to the desired protein at its C terminus, followed by a generic terminator and a selection marker. Primers contain flanking sequences directing the tagging cassette to recombine into the gene end, creating a fused protein.
Templates used for C’ tags should encode a stop codon at the end of the tag’s sequence, and a terminator for transcription termination before the selection cassette.
Forward primer: 40bp before the gene’s Stop-codon (excluding the Stop-codon from the primer), followed by the “forward primer” sequence of the transformation cassette (plasmid dependent).
N’ tagging primers are intended to amplify a cassette containing a selection marker followed by a promoter and a tag to be fused to the desired protein at its N terminus. Primers contain flanking sequences directing the tagging cassette to recombine into the gene start, creating a fused protein.
Templates used for N’ tags should contain a selection marker followed by a promoter for transcription, and a start codon for the tag to ensure initiation of translation of the fused protein.
Forward primer: 40bp upstream to the gene’s start codon (ATG) (including the ATG in the primer), followed by the “forward primer” sequence of the transformation cassette (plasmid dependent).
Reverse primer: The reverse complement of 40bp after the gene’s start codon (ATG) (excluding the ATG from the primer), followed by the “reverse primer” sequence of the transformation cassette (plasmid dependent).
To verify correct insertion use N’-tag Check primers.
Check primers are intended for verification of correct insertion of the transformation cassettes to the desired location (gene).
Click here for a full description of all check primer types in the information page.
Primers were generated using BatchPrimer3 and are on average 20bp long, have 40% GC content, and contain a one bp GC clamp (at least one G or C at the 3’ end). Primers have an average melting temperature (Tm) of 60°C, and we recommend setting this as the PCR annealing temperature, as we successfully tested 129 primers so far with this parameter using various polymerases.
Note – following transformations yeast often become aneuploid and so the mere presence of a selection cassette in your gene’s locus does not verify that all copies of your gene have been tagged or deleted. Therefore it is recommended to use several phenotypic and PCR verification strategies (such as “WT check” and "UTR check") for gene deletions and tagging.
If a custom plasmid not represented in Primers-4-Yeast is required, users should input the plasmid name and cassette amplification sequences (forward and reverse primer sequences), and select the gene targeting types. Primers-4-Yeast will design primers for amplification of the cassette from the custom plasmid, and their insertion into each ORF from the input list, based on the targeting type.
