Plan up to 10 Sanger primer pairs and overlapping primer-walking reads for a selected DNA region. Set expected read length and low-quality edges, and mark mutation positions to check their predicted coverage.
Download primers as FASTA or a TSV table, and save the complete coverage map as SVG or PNG. Map downloads include the displayed read zones beyond the visible scroll area.
Useful Checks
The tool plans multiple Sanger primer zones from amplicon size, expected read length, and the reliable window after low-quality edges are removed.
Listed mutation positions influence zone planning and are checked for forward and reverse support inside reliable read regions.
Generated primer pairs can be sent to Primer Binding Checker to verify exact binding positions, orientation, and expected product size on the same template.
Minimum Inputs Needed
The sequence of the DNA you will submit for sequencing: a plasmid or the actual purified PCR product.
Amplification region start, using 1-based coordinates in the pasted template.
Amplification region end, included in the region. Include flanking sequence only if it is present in the DNA being sequenced; a purified PCR product does not contain the original template's outside flanks.
Optional Settings
Mutation positions (optional)
Enter comma-separated, 1-based positions in the full template, not relative to the selected region. These mark bases to check in a read; they do not introduce mutations.
Expected read length, bp
Use the read length you expect from your sequencing provider.
Low-quality edge per read, bp
The same number of bases is excluded at both ends of each planned read. A 650 bp read with 50 bp edges gives a 550 bp usable window. This edge value also sets the minimum overlap between neighbouring reliable read windows. Review the map for gaps; real read quality may differ.
Read zones / primer pairs
Leave this blank for automatic planning of up to 10 pairs, or enter a whole number from 1 to 10. If the suggested count exceeds 10, automatic planning asks for a smaller region. A manual count below the suggestion can leave coverage gaps.
Target primer Tm, deg C
The preferred melting temperature. The tool searches binding lengths from 18 to 32 bases; it may not reach the target exactly.
Na+, Mg2+, primer, and dNTP settings
Use your sequencing provider's reaction conditions. Na+ equivalent, Mg2+ and total dNTP are in mM; primer concentration is in nM. For example, 0.2 mM of each dNTP is 0.8 mM total.
How To Use
Paste or upload the template sequence.
Enter the amplicon start and end.
Add mutation positions if you need to verify specific bases.
Leave Read zones / primer pairs blank for automatic planning within the 10-pair limit, or enter 1–10 manually. For longer targets, design smaller overlapping regions.
Click Design Sanger primers.
Changing design inputs clears the old plan. Click Design Sanger primers again to update it.
Understanding The Results
The coverage map shows predicted usable read windows under your selected read-length and edge settings.
The mutation table shows whether each listed position falls within predicted forward and reverse read windows.
A planned pair represents two separate Sanger sequencing reactions, each with one primer. Do not combine forward and reverse primers in one standard sequencing reaction.
The primer table gives primer sequence, Tm, GC%, binding position, and read span.
Copy all primers as FASTA or copy the primer table for ordering and records.
Use the transfer button to check every generated pair in Primer Binding Checker. The complete design fits its 10-pair limit.
Send results to Primer Scan (Beta) to screen the named primer pairs against a separately chosen reference. No template DNA is sent. A Sanger sequencing reaction still uses one primer.
Boundary and coverage warnings identify short flanking sequence, uncovered regions, or plans below the suggested zone count.
Accepted Input Formats
Raw DNA sequence.
FASTA.
GenBank text with an ORIGIN sequence section; feature annotations are ignored.
Multiple FASTA records are concatenated into one template. Analyze one record at a time to avoid artificial junctions.
.dna / SnapGene binary files are not supported. Export as FASTA or GenBank.
Mutation positions are 1-based numbers separated by commas.
Assumptions And Limitations
Real Sanger read quality depends on template quality, primer quality, and provider conditions.
Read windows are planning assumptions, not measured chromatogram quality. This tool does not read trace files or call mutations.
This planner treats the sequence as linear. For a plasmid target crossing base 1, rotate the sequence text so that target and its flanks are contiguous, then update coordinates. This changes the sequence representation; it does not require physically cutting the plasmid.
Primer Tm and structure checks are screening estimates; external manufacturer calculators are available for final protocol-specific review.
The browser tool accepts sequence files smaller than 2 MB and templates up to 250,000 bp. Each design is limited to 10 pairs; template capacity does not imply complete sequencing coverage. With 650 bp reads and 50 bp low-quality edges, ten planned zones span approximately 5,050 bp. With 700 bp reads, they span approximately 5,550 bp under the same edge settings. Primer placement and available flanks can still leave gaps; review the coverage map.
Example
For a 1600 bp amplicon with mutations at 430 and 1040, enter the region, list the mutations, use the suggested count, and check that each mutation has reliable forward and reverse coverage.
Use this result with other tools
Open in Primer Binding Checker: sends the template, primer-pair names and full primer sequences to Primer Binding Checker for exact mapping. All generated pairs, up to 10, are transferred together. Run Check primer binding in the receiving page.
Send results to Primer Scan: sends all generated pairs, up to ten named pairs to Primer Scan for reference screening. The template is not sent; select the host or custom reference there. Confirm the imported names and run the scan.
These pairs organize forward and reverse sequencing coverage. Sanger reads normally use one primer per sequencing reaction. A product predicted by a pair-based checker does not turn the sequencing plan into a PCR protocol; inspect each primer’s mapping for the intended sequencing reaction.
Both receiving tools retain the original Sanger names beside their local pair labels. Up to 10 pairs can be generated and transferred together without shortening any primer. Transfers stay in this browser, expire after 30 minutes and can be imported once.
Correcting highlighted input
When the main action rejects pasted sequence text, unsupported characters are highlighted in the field and the error message explains the problem. Correct the marked characters using the alphabet allowed by this tool, then run it again. The highlights update as you edit. A malformed FASTA or GenBank record may require a structural correction even when no individual character is marked.
Use note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.