Yeast cloning batch primer design

Design PCR-based cloning strategies for yeast in batch.

Powered by OpenCloning. To learn more about all OpenCloning features, see docs.opencloning.org. If you have feedback, reach out to manuel.lera-ramirez@ucl.ac.uk.

How to use

You can check out this video for a quick overview.

• Select the genome assembly
• Select the cloning type
• Enter yeast systematic gene IDs (e.g. SPBC15D4.01c).
• Select a marker plasmid.
• Specify verification primers.
• The pipeline designs primers for integration and checking.
• You can decide if you just want the primers or if you want to simulate the cloning.

What it returns

Primers only

Shows you a table with the primers.

Simulate cloning

After a delay (can be long), it returns a ZIP archive with:

• Per-gene folders that contain:
  • Genbank files with the sequences involved
  • A json file containing the entire cloning strategy. You can drag-and-drop it into the OpenCloning website to visualize the strategy. It will look similar to this.
• Summary tables (TSV, Excel, YAML)

Best way to get started

Just try with the below example, it will return a zip file and you can drag-and-drop the json file SPAPB1A10.09/cloning_strategy.json into OpenCloning to visualize the strategy. Check also the file summary.xlsx file to see the primers, and the multiple genbank files with the sequences involved, you can open those in SnapGene, Benchling, etc.

Genome assembly

pombe cerevisiae custom

Interested in supporting more species or genomes? Reach out to manuel.lera-ramirez@ucl.ac.uk.

Cloning type

Gene deletion Gene C-term tagging Promoter insertion (no tag) Promoter insertion (with tag, GFP) Promoter insertion (with tag, 3HA) Promoter insertion (with tag, GST)

Gene list (one gene per line)

Yeast systematic IDs only (e.g. SPAPB1A10.09, for pombe, YOR058C for cerevisiae). One gene ID per line; empty lines are ignored.

SPAPB1A10.09 SPBC15D4.01c

Plasmid primer binding Restore defaults

Primer part that binds to the plasmid. By default, values for pFA6a-derived plasmids are used.

Forward primer binding

Reverse primer binding

Desired output

Design primers and simulate cloning Design primers only

Plasmid source

The plasmid supplies the marker cassette (e.g. KanMX6, NatMX6) to be PCR-amplified and integrated into each gene locus via homologous recombination.

Example plasmid (pFA6a-kanMX6, pFA6a-natMX6, pFA6a-hphMX6) Provide Addgene Identifier Upload Plasmid File

Upload plasmid sequence file

Accepted formats: GenBank (.gb, .gbk, .genbank), FASTA (.fasta, .fa), or snapgene (.dna).

Addgene identifier

Numeric Addgene ID of the plasmid (e.g. 39296 for pFA6a-KanMX6). Find IDs at addgene.org.

Resistance marker

Choose KanMX6, NatMX6, or HphMX6 for standard cassettes, or "Other". Cassette checking primers are optional: provide forward only, reverse only, both, or neither to control which verification PCRs are simulated. Standard cassettes are pre-filled automatically.

KanMx6 NatMx6 HphMx6 Other

Checking primer (forward)

Aligns to the top strand of the inserted cassette.

Checking primer (reverse)

Aligns to the bottom strand of the inserted cassette.

Submit to run the pipeline. You will receive a ZIP file containing per-gene folders (primers, cloning strategy, GenBank sequences) and summary tables (TSV, Excel, YAML).

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