DNA to Protein Translation — Six-Frame Online Tool

Translates a nucleotide sequence into amino acids using the standard genetic code (NCBI translation table 1). Each triplet of bases forms a codon; 64 codons encode 20 amino acids plus 3 stop signals.

Why six frames? For an unknown sequence, you do not know which reading frame is used, or whether the coding sequence lies on the forward or reverse-complement strand. Three offsets (0/1/2) on the forward strand plus three on the reverse complement give six possibilities; checking all six ensures nothing is missed.

The tool also reports every open reading frame (ORF) in each frame: any stretch that begins with ATG, ends at a stop codon, and meets the minimum-length threshold. This is the simplest ORF definition. Because eukaryotic genes contain introns, this approach does not apply to eukaryotic genomic DNA — use it for prokaryotic genes, cDNA, and plasmid sequences.

Finding an ORF is not the same as finding a gene

This is the step most often glossed over when using six-frame translation. For an ORF to actually be expressed, it also needs:

• A promoter — RNA polymerase must be able to transcribe this region
• A ribosome-binding site — the Shine-Dalgarno sequence upstream of the ATG in prokaryotes, or the Kozak consensus context around the ATG in eukaryotes
• Introns in eukaryotes — ORFs in genomic sequence are interrupted by introns, so use cDNA rather than genomic DNA when searching for eukaryotic ORFs

The longest ORF is not necessarily the real one. Long ORFs can appear by chance in random sequence: with four bases at equal frequency, each codon has a 3/64 probability of being a stop codon, so a stop is encountered on average every ~21 codons. The variance is high, however, and runs of over a hundred stop-free codons occur by chance. Confirming a real gene requires the regulatory elements above, homology evidence, and expression data — length alone is insufficient.

The start codon is not always ATG

The NCBI standard genetic code designates TTG and CTG as alternative start codons, and GTG is used as a start in rare cases. Nearly all eukaryotic genes begin with ATG, but restricting to ATG alone when annotating prokaryotic genomes will miss real genes. This tool uses the simplest ATG-only definition for ORF finding — keep this in mind for prokaryotic sequences. All 27 genetic code tables list the start codons for each table.

Consequences of using the wrong genetic code

Mitochondria, certain ciliates, and mycoplasmas do not use the standard code. In vertebrate mitochondria, for example, TGA is a stop codon in the standard table but encodes tryptophan in the mitochondrial table — translating with the standard table will incorrectly truncate the protein at the first TGA. Before analyzing non-nuclear genomic sequences, confirm which table applies.

On the minimum ORF length

A threshold of 100 codons (300 bp) is common practice for filtering random-noise ORFs. This is an engineering convention, not a biological boundary — genuine short-peptide genes of only a few dozen amino acids will be discarded at this threshold. When studying small or regulatory peptides, lower the threshold and accept the increased false-positive rate.