DNA Sequence

Raw sequence or one or more FASTA records. Input limit: 10,000,000 characters.

Parameters

Restriction enzyme

Bases per line

Show translation for

Genetic code

💡 Quick Summary

Mutate for Digest accepts a DNA sequence and searches for regions that can be converted to a chosen restriction site by introducing one or two silent nucleotide substitutions. Protein translations are shown alongside so you can immediately see which reading frames, if any, are altered by each proposed mutation.

📋 How to Use

Paste a raw DNA sequence or one or more FASTA sequences into the textarea. Input limit: 10,000,000 characters.
Select the restriction enzyme you want to introduce from the dropdown (default: BamHI).
Choose how many bases per line to display (default: 60).
Choose which reading frame(s) to show translations for, or select "none" to skip translation.
Select the appropriate genetic code (default: standard / transl_table=1).
Click Run. The output shows the original sequence (grey) and mutated version (amber) side-by-side with restriction site markers. Each mutation requires ≤ 2 base changes.
Click Load Example to try with a sample CDS sequence.

🧮 Formulas & Logic

Single-N variants

For each non-N position in the site pattern, one variant is generated with that position replaced by N (any nucleotide)

Double-N variants

For sites longer than 6 bp, all pairs of non-N positions are also replaced (two N substitutions)

Match filter

Any "mutated" site that coincides with an existing real site at the same position is discarded

Overlap filter

Overlapping mutated sites are deduplicated to prevent one mutation from altering another site

📊 Result Interpretation

Amber (mutated) row

The sequence as it would look after introducing the minimum substitutions needed to create the restriction site

Grey (current) row

Your original input sequence at the same positions

Site label (amber)

Enzyme name and 1-based position of the cut site in the mutated sequence

Site label (grey)

Enzyme name and position of any sites already present in the original sequence

Translation rows

Protein translation of the reading frame(s) chosen — check that the amber and grey rows match to confirm the mutation is silent

🔬 Applications

Designing site-directed mutagenesis primers that introduce a useful restriction site into a coding sequence
Identifying silent mutation positions for restriction-enzyme-based cloning (RE-cloning)
Checking whether a desired restriction site can be created without changing the encoded protein
Locating the closest restriction site to a specific codon for diagnostic purposes

⚠️ Common Mistakes & Warnings

Mutations are not guaranteed to be silent

The tool identifies positions where the DNA can be changed to create the site, but does not verify that every substitution preserves the amino acid. Always compare the amber (mutated) and grey (original) translation rows. If they differ, the mutation changes the protein sequence.

Input limit is 10,000,000 characters

For very long sequences the search may be slow. For PCR primer design purposes a window of a few hundred bases around the target codon is usually sufficient.

❓ Frequently Asked Questions

What does "N" mean in the mutated pattern?

N stands for any nucleotide (A, T, G, or C). When the tool generates degenerate site variants, it replaces one or two fixed positions with N, then searches your sequence for a match. Any base found at an N position is left unchanged — only the other positions in the site are mutated.

Why are some candidate mutations not shown?

Candidates are removed if (1) the site already exists naturally at that position, or (2) two overlapping candidates would interfere with each other — only the first is kept.

How do I verify the mutation is silent?

Compare the amber (mutated) translation row with the grey (original) row for the reading frame your gene uses. Identical letters at every position means the amino acid sequence is unchanged.