Use this to design sequencing primers across an amplicon. The tool plans around the useful part of a Sanger read, not only the full read length.
Useful Checks
Generated Sanger primer pairs can be sent to Primer Binding Checker for binding-position validation on the same template.
The Sanger tool can prioritize listed mutation positions while choosing read zones, so important variants are kept inside reliable read regions rather than near read edges or junctions.
The tool can plan multiple Sanger primer zones from the amplicon size, expected read length, and reliable read window.
If you enter mutation positions, the result checks whether those bases sit inside the reliable part of the read, not only near an edge.
Use Primer Binding Checker after design to catch primers that bind in the wrong orientation or give an unexpected product size.
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
More zones give more coverage. Use the suggested count if unsure.
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.
Use the suggested primer-pair count unless you already know how many primers you want.
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.
Use the transfer button to check the designed primers in Primer Binding Checker.
Accepted Input Formats
Raw DNA sequence.
FASTA.
GenBank where supported.
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.
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.