1 Sequence annotation
Locates your amplicon in the human genome (GRCh38) and returns the gene, its MANE Select transcript and the sequence with exons in UPPERCASE and introns in lowercase.
Paste the amplicon from your in-silico PCR (a FASTA header line is fine). The reference index covers the HFE locus (chr6:26,077,000–26,108,000).
2 Translation in six frames
Translates a DNA sequence in the three forward and three reverse reading frames. Stop codons are shown as *.
Think: which part of the Tool 1 output should you translate? Which frame is the correct one? (The exon you amplified is part of the CDS.)
3 Alignment to the reference protein
Aligns your translated peptide to the full reference protein from UniProt. Each line starts with the reference position of its first residue, so you can locate the cysteine at position 282 (C282).
Find the human HFE protein on UniProt and copy its accession code.
4 3D structure: locate a subsequence
Loads a structure from the PDB and highlights a stretch of amino acids in red, with side chains and labels. Labels use UniProt numbering (structures are often numbered differently).
You need the PDB code of HFE in complex with the transferrin receptor, and the UniProt accession from Tool 3. Tip: copy a few residues around C282 from the Tool 3 alignment.
5 Disulfide bridges
Lists every cysteine of the reference protein and whether its sulfur atom (SG) is bonded to another cysteine in the structure (S–S distance ≈ 2.05 Å). Click Show to view the bridge in 3D.
Uses the structure loaded in Tool 4.
6 Codons to amino acids
Once we have the chromatograms, we need to know where to look. This tool shows which codon encodes each amino acid, and the position of the codon in the sequence you paste.
Paste the exonic part of the amplicon and pick the reading frame you chose in Tool 2. Find C282 using the neighbouring residues from Tool 3.
7 Chromatogram viewer
Open a Sanger sequencing file (.ab1). Search for the bases around the C282 codon to jump to it, then look at the peaks:
one clean peak means homozygous; two overlapping peaks of different colours at the same position means heterozygous.
Files are read in your browser; nothing is uploaded.