For academic use only.
For commercial use, please contact Nooriel Banayan (, John F. Hunt (, or Columbia Science and Technology Ventures (

Bulk Lysine-To-Arginine Mutagenesis

K to R substutions
D to E substutions
N to Q substutions

Once you’ve submitted, the screen will be blank while the calculations run. This is normal and intended, do not panic. Calculations take ~2min for a 120-residue protein with 5000 homologs.

Input Information

ALN File:
  • Alignment File in CLUSTAL format (.aln)
    • Generate an .aln file by submitting your .fasta file with your protein sequences to the CLUSTAL website for alignment.
Reference ID:
  • The ID of your reference sequence EXACTLY as it appears in the .aln file.
    • The reference ID is the ID of your reference sequence in your alignment file. For example, your reference ID would be "kinase" if the header line for your target sequence is "kinase". Please make sure your reference ID does not have any spaces.
  • The threshold value specifies the minimum expectation value for independent observations of the intended substitution in the seven most diverged sequences required for a candidate site to get a high-confidence recommendation for mutation (default = 1.1, must be greater than 1).
Run time:
  • Run-time for the analysis is approximately 2 minute for a 120-residue target sequence with 5,000 homologous sequences in the input file.