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. For best Sanger primer placement, include at least 100 bp upstream and 100 bp downstream of the amplification region of interest.

Read planning

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.
PCR settings

References

References for Sanger sequencing primer design: chain-termination sequencing context, primer walking, nearest-neighbor primer Tm calculations, salt and magnesium correction, and primer-quality screening. Mol Biology Tools implementation code is original.

  1. Sanger sequencing method context: Sanger, Nicklen, and Coulson (1977), chain-termination DNA sequencing.
  2. Sanger primer Tm calculations: SantaLucia J. (1998), unified nearest-neighbor DNA thermodynamics.
  3. Salt and magnesium correction for DNA duplex Tm: Owczarzy et al. (2008), DNA duplex stability in magnesium and monovalent cation solutions.
  4. Primer-quality screening heuristics: Untergasser et al. (2012), Primer3 design constraints and checks.