- 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.)
Primer design and other tools
Inserting a specific sequence in any vector
The following protocol walks you through the steps for designing PCR primers to seamlessly insert a sequence, such as a small tag or domain, in any vector with In-Fusion Cloning. Use our online primer design tool to:
- add the vector sequence of your choice,
- specify the exact nucleotides to be added,
- choose the insertion locus (independent of restriction sites), 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. Choose insertion location
- 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 the exact location where you want to insert the new sequence. Ensure that a black line appears in the correct spot.
D. Enter insertion sequence
- In the "Select Modification" box, click the [Insert] button.
- Enter your desired insertion sequence 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 region and sequence for insertion.
E. Design primers
- When you are satisfied with your modification selection, click on the [Design Primers] button.
- View your results. The window will switch from the Design Page tab to the Results Page tab. This tab is divided into the following sections: your final vector map/sequence and an output protocol with oligo and PCR information (see Step 3).
- Download your primer information as an Excel file by clicking the [Download Results] button at the bottom of the page. This data can be entered into any nucleotide sequence analysis software tool, such as SnapGene Viewer (available for free online), which allows easy visualization of In-Fusion Cloning primer locations in both sense and antisense strands.
Seamless cloning primer design tips
Useful tips to keep in mind when designing your seamless cloning projects.
In-Fusion Cloning primer design tutorials
Learn how to use our online tool to quickly and easily design primers for every In-Fusion Cloning project.
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