Simulate In-Fusion® Cloning with One Insert
How do I simulate In-Fusion® cloning in Snapgene?
In-Fusion® is a registered trademark of Takara Bio Inc, USA.
See the Takara website for more information about In-Fusion® cloning – https://www.takarabio.com/products/cloning/in-fusion-cloning.
To see a video on simulating In-Fusion® cloning in SnapGene follow this link.
In this lesson we will insert a coding sequence region (CDS, excluding signal peptide and stop codon) into pET-26b(+). The xynB CDS will be positioned so that it is in-frame with the vector-based N-terminal PelB leader sequence and in-frame with the vector-based C-terminal HIS-tag.
Open the Vector Sequence
Import or open the plasmid vector sequence that will be used for In-Fusion cloning.
- Click within the sequence to select the insertion point, or
- Click on an enzyme site to select it as the insertion point, or
- Select a specific region that will be replaced by the insert, or
- Select a restriction fragment that will be replaced by the insert.
To select a restriction fragment for replacement, click on the first restriction site, then shift click on the second restriction site. Alternatively, click on the first site then drag to the second site.
In the above example the pET-26b(+) plasmid linearized with XhoI and NcoI will be used as vector for In-Fusion Cloning.
Start the In-Fusion® Cloning Tool
Click Actions → In-Fusion® Cloning → Insert Fragment.
The In-Fusion® tool opens showing the "Vector" tab, with the option to "Linearize with restriction enzymes" automatically selected and the appropriate enzyme fragment for replacement selected.
When starting the In-Fusion® Cloning tool, the site or DNA sequence selection in the foremost window will be automatically set as the vector region for insertion/replacement by In-Fusion® Cloning.
If a vector sequence is not open when you start the In-Fusion® cloning tool then you can choose and define the vector in the Vector tab.
Check the Vector Orientation
If required, set the "Orientation of Vector" reverse orientation for the vector.
In the above example, viewing the sequence in Map view and zooming in, we see the pET-26b(+) pelB-MCS-HIS-tag Features are in the reverse orientation (1). Therefore, we set the "Orientation of Vector" to reverse (2).
Specify the Insert Fragment Source Sequence
Switch to the "Fragment" tab.
There are a number of ways to specify the fragment source sequence, you can use any one of the following methods:
- Click the dropdown to set the "Source of fragment", or
- Click the "Click here" link to browse and set the "Source of Fragment".
Drag and Drop
Drag and drop a sequence file into the Fragment panel.
Cut and Paste
Copy a sequence from any compatible source file and paste to import it as the fragment source.
Define the Insert Region
In Map or Sequence view, select the region of the fragment sequence to be inserted into the vector.
In this example we manually define the insert region, starting at the boundary with the XynB signal peptide and excluding the xynB stop codon to allow fusion to a vector-based C-terminal HIS-tag.
Switch to Sequence view, locate the start of the insert and click to set the cursor to the start position.
Scroll to the end of the insert and SHIFT-click to select the insert region.
Design Primers to Amplify the Insert Fragment
In this example we will regenerate the vector-based XhoI and NcoI restriction sites.
Switch to the Product tab and click Choose Overlapping PCR Primers.
Set the desired Target Tm for the primers and the desired overlap length.
Click Choose Primers to design new PCR primers.
Validate the Vector/Insert Fusion Points
In the Product tab, switch to Sequence view.
In this example we observe that the vector-based pelB signal sequence is not in-frame with the fragment-based xynB CDS.
To create an in-frame fusion, addition of two further nucleotides between the regenerated NcoI site and the xynB CDS is required. To do this we will extend the insert fragment at the 5' end by 2 nucleotides, and redesign the primers.
Switch back to the Fragment tab, in Sequence view, add two nucleotides to the 5' end of the insert portion of the primer.
In this example the vector-base G is combined with the additional nucleotides "ct" to add an Alanine (GCT codon) between the vector-based pelB and the xynB CDS.
Switch to the Product tab, Sequence view and confirm an in-frame fusion has been created.
The xynB CDS is now observed to be in-frame with the vector-based pelB signal peptide, and the NcoI site has been regenerated.
Scroll to the end of the insert fragment and confirm the C-terminal fusion.
The xynB CDS is observed to be in-frame with the vector-based 6-HIS tag, so no primer-based adjustment of the fusion is required.
Note the XhoI site has been regenerated.
Name the New Primers and Create the Product Sequence
Switch to the Fragment tab.
Enter appropriate names for the two new primers.
Enter an appropriate name for the product.
Click Clone to create the new In-Fusion product sequence.
Click File → Save to save the new sequence to an appropriate location on your computer.
View the newly created product file, switch to History view to see all steps simulated during the In-Fusion cloning procedure.
Order the PCR primers
See Export Primers for details on how to order the new primer sequences.