- Change the selection to 'Mutagenesis' in the "Select Project Type" box in the left column. ('Cloning' will be selected by default upon opening the primer design tool.)
Deleting and replacing a sequence in any vector
The following protocol walks you through the steps for designing PCR primers to delete a sequence and simultaneously replace it with a different sequence in any vector, with just one round of In-Fusion Cloning. This process is ideal for domain swapping studies. Use our online primer design tool to:
- add the vector sequence of your choice,
- specify the exact nucleotides to be deleted,
- enter your replacement sequence, and
- download primer and PCR information based on your design.
A. Select project type
B. Input vector sequence
Use the "Add Vector To Mutagenize" box to input your vector sequence in one of the following ways:
Provide your own vector sequence
- Click on 'Input sequence'.
- Paste your nucleotide sequence into the text box as plain text, without nucleotide or line numbering.
Choose from the preloaded list of vectors
- Click on the plus sign next to "Select a Takara or Clontech Vector".
- Choose a vector from the list.
C. Select sequence to replace
- In the pane on the right side of the screen, navigate to the area you want to modify by clicking on the vector map image to jump to a general area or feature.
- Use the nucleotide view to select and highlight the exact sequence you wish to delete.
D. Enter replacement sequence
- In the "Select Modification" box, click the [Replace Selection] button.
- Enter the sequence to replace the selected area into the box that appears on top of the nucleotide view of the vector and press ENTER on your keyboard.
- Double-check that your construct map and sequence show the correct deletion and replacement sequence.
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