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Design from an amplicon FASTA

Use FASTA mode when each record contains a locus or expected amplicon and the required interval is centered within that record.

Run the example

From the repository root:

smfprimer design fasta docs/examples/amplicon.fa \
  --target-width 10 \
  --workflow conversion \
  --context both \
  --converted-strand top \
  --product-size 50:100 \
  --min-tm 30 \
  --optimum-tm 50 \
  --max-tm 80 \
  --output primers.tsv

The relaxed Tm range makes the short repetitive documentation fixture useful; use assay-appropriate values for real designs.

Inspect the result

Open primers.tsv and begin with:

  • status: ok means Primer3 returned a pair.
  • rank: lower ranks have lower Primer3 pair penalties.
  • forward_order_sequence and reverse_order_sequence: final 5′→3′ oligos.
  • forward_degeneracies and reverse_degeneracies: IUPAC ambiguity counts.
  • amplicon_start, amplicon_end, and amplicon_length: predicted product.

Open primers.gb in a GenBank viewer to see the required interval, amplicon, and primer directions together.

Multiple records

Every FASTA record produces its own outcome. Failed records remain in the table, making a batch run auditable.

Add --gtf annotations.gtf.gz when FASTA record names and coordinates match the supplied annotation. Overlapping GTF features are included in the GenBank companion.