Universal primer design on MSA

The tool performs a multiple sequence alignment, derives a per-column conservation profile, and designs universal primers anchored on conserved regions.

Every primer's 3′-end is guaranteed conserved across all sequences. The 5′-end may sit on variable columns (rendered as IUPAC-degenerate bases) when “Allow degenerate 5′-end” is enabled; otherwise primers are fully conserved.

Upload local FASTA file — plain FASTA or multi-FASTA, ≥2 DNA sequences

Upload FASTA:

Universal Primer Options

Length range (nt)

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Tm range (°C)

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Product size (bp)

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Conserved 3′-window (nt)

Min. linguistic complexity (%)

Max. pair Tm difference (°C)

Gap open penalty

Gap extend penalty

Max forward sets to report

Allow degenerate 5′-end (variable columns become IUPAC codes; 3′-end stays 100% conserved)

Report overlapping primers (off: keep only the best primer by linguistic complexity in each region)

Auto-correct sequence orientation (reverse-complement) before alignment

How it works. Coordinates in the output are 1-based alignment columns. A primer is reported only when its 3′-anchor window is 100% conserved across every input sequence. Product size is reported as the range of ungapped amplicon lengths over all templates.
Exporting results: click inside a result area → Ctrl+A → Ctrl+C, or use the Copy / Download buttons.