Sanger Primer Designer

Plan primers across an amplicon with this free online Sanger sequencing primer design tool, using reliable read windows, mutation positions, primer walking, and downstream binding-position validation.

Sanger primer designer inputs
No sequence loaded.

Accepted file types: .txt, .fa, .fasta, .fna, .ffn, .seq, .gb, .gbk, .genbank. Plain-text DNA, FASTA, or GenBank text only.

Circular DNA/plasmids are not handled as circular yet. For regions that cross base 1, paste the relevant region as linear FASTA or raw DNA sequence.

Amplification region

Use 1-based coordinates. Include at least 100 bp upstream and downstream for primer placement only if those flanks are present in the DNA you will submit for sequencing. A purified PCR product does not contain flanks outside that product.

Read planning

Plan 1–10 pairs per design. Leave the count blank for automatic planning within this limit. For longer targets, choose smaller overlapping regions and review coverage warnings.

Each read zone generates one forward and one reverse sequencing primer. The low-quality edge is also used as the minimum reliable-window overlap, so fuzzy read ends are covered by reliable sequence from the next read.

Load a sequence to see read-planning suggestions.
Tm calculation settings

References

Sources for Sanger sequencing, primer Tm, and sequence formats. Read-window planning and primer-quality screening use original Mol Biology Tools code, not the Primer3 engine.

  1. Sanger sequencing method context: Sanger, Nicklen, and Coulson (1977), chain-termination DNA sequencing.
  2. Perfect-match primer Tm: SantaLucia (1998), nearest-neighbor DNA thermodynamics. Includes terminal initiation and self-complementarity corrections.
  3. Salt and magnesium correction: Owczarzy et al. (2008), mixed-salt duplex stability. Free Mg2+ is approximated as total magnesium minus total dNTP, with a minimum of zero.
  4. Primer-design background: Untergasser et al. (2012), Primer3. Background reading, not the implementation used here. Predicted read coverage and structure scores are planning estimates.
  5. Sequence-file formats: NCBI FASTA documentation and GenBank sample record.