Use this free online Sanger sequencing primer design tool to plan primers across an amplicon using reliable read windows, mutation positions, and primer walking rather than full read length alone.
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
DNA sequence.
Amplification region start.
Amplification region end.
Optional Settings
Mutation positions (optional)
Enter positions you care about. The tool checks whether these bases fall inside reliable read regions.
Expected read length, bp
Use the read length you expect from your sequencing provider.
Low-quality edge per read, bp
Bases at the start and end of a read are often less reliable. This setting trims them during planning.
Read zones / primer pairs
Leave this blank for the automatically suggested count. Enter a whole number only when you want to override the plan manually.
Target primer Tm, deg C
The preferred Tm for designed primers.
Na+, Mg2+, primer, and dNTP settings
These change the Tm estimate. Defaults are suitable for most planning.
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 to use the suggested automatic count, or enter a manual count.
Click Design Sanger primers.
Understanding The Results
The coverage map shows which parts of the amplicon are covered by reliable reads.
The mutation table shows whether each mutation has forward and reverse read support.
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 the designed primers in Primer Binding Checker. If more than 15 pairs are generated, only the first 15 are transferred.
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.
The read length and low-quality edge are planning assumptions.
If a mutation is close to a read edge, add more primer pairs or adjust manually.
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.
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 note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.