Welcome to PRIMUTANT

This is a FREE and SIMPLE program that designs primer pairs used to create arrayed mutagenesis libraries for your protein of interest. The designed primers are optimized for use in our high-throughput cloning pipeline paper published at STAR Protocols.

Please follow the step-by-step instructions below to generate a downloadable .csv file of custom mutagenesis primers. The .csv file can be readily used to order pre-mixed primer pairs from commercial oligo synthesis providers.

Step 1: Filename and organism

Filename (without special characters or spaces):


Organism Classification:



Step 2: Sequence

Please enter the nucleotide sequence from your construct corresponding to the protein coding sequence. Include at least 30 nucleotides upstream and downstream of the corresponding residues being targeted (Note: this may necessitate including vector sequences outside the coding sequence).


Step 3: Target Region Determination

(a) Please input the position of the first nucleotide of the start codon. Note that the first nucleotide of the inputted sequence equals position 1. For example, if the inputted sequence was "TGGATCAUGTTGACTT..." you would enter 7 in the box below.


(b) Please input the starting and ending positions of the nucleotides for the region to be mutated.

Start:

End:

Step 4: Mutation Choice

Choose between 3 options:

  • Site Saturation - Mutates each target residue to the other 19 amino acid possibilities
  • Phosphorylation Site - Mutates each serine and threonine residue target to glutamate (phosphomimetic) or alanine (phosphorylation deficient)
  • Scanning - Mutates each target residue to the amino acid indicated


For scanning mutagensis ONLY: Choose the amino acid you wish to mutate the WT target residue to.


For phosphorylation site mutagenesis ONLY: Choose the type of mutagenesis.





Once the submit button is pressed, the list of primers will be saved in a .csv file.