Simulate In-Fusion® Cloning
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 "clone" the coding sequence region (CDS) of xynB (excluding signal peptide and stop codon) into pET-26b(+). The xynB CDS will be positioned in-frame with the N-terminal vector-based PelB leader sequence and in-frame with the C-terminal vector-based HIS-tag.
Summary of steps in this lesson:
1. Define the pET26b(+) NcoI-XhoI restriction fragment used for the In-Fusion® reaction.
2. Define the xynB insert fragment sequence for PCR amplification and subsequent In-Fusion cloning.
3. Have SnapGene design primers for PCR amplification of the insert with extensions to create overlap with the vector NcoI-XhoI fragment. The primers will also regenerate the NcoI and XhoI sites.
4. Simulate the In-Fusion® reaction between the vector and overlapping insert fragment.
Open and Prepare a Cloning Vector Sequence
Import or open a sequence file for the plasmid vector that will be used for In-Fusion cloning.
In this example we will download the pET-26b(+) vector from the SnapGene "Online Sequence Library".
Open or Import a Vector Sequence
Open a vector sequence file, or to import a vector from the SnapGene Online Library click File → Import → SnapGene Online Sequences.
Search for pET-26 to find the file, select the file in the list, and click Import to download and open the sequence as a new file.
Check the Vector Orientation
Switch to Sequence view and locate the multiple cloning site (MCS). In this example vector, the MCS and expression elements (pelB signal peptide and HIS-tag) are in reverse orientation.
Click menu View → Flip Sequence to reverse complement the pET-26b(+) vector sequence. This will simplify the In-Fusion® cloning process.
Click File → Save to save the pET-26b(+) vector to an appropriate location on your computer.
The pelB signal sequence feature now runs in the forward direction.
Define the Vector Region for Replacement
Select the restriction fragment for replacement by the insert. In this example we select the NcoI site label, then shift click on the XhoI site label to select the region.
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 fragment for replacement already selected.
Select an Insert Fragment
Switch to the "Fragment" tab, set the "Source of Fragment" from the dropdown menu, or click Browse to locate the insert sequence file on your computer.
In this example we will 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 desired insert region.
Design Primers to Amplify the Insert Fragment
Switch to the Product tab and click Choose Overlapping PCR Primers.
Set the desired Target Tm for the primers and the desired overlap length.
In this example we will choose to regenerate the vector NcoI and XhoI restriction sites, check the options to regenerate the sites.
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.
Observation indicates that 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, select the revised insert region, this time including two nucleotides upstream of the signal peptide boundary .
Switch to the Product tab and repeat the step Choose Overlapping PCR primers, make sure you choose to regenerate the NcoI and XhoI sites.
Switch to Sequence view, view the fusion point, the pelB signal peptide is now in-frame with the xynB CDS.
Note the NcoI site has been regenerated.
Scroll to the end of the insert fragment, the xynB CDS is observed to be in-frame with the vector-based 6-HIS tag, so no further adjustment of the insert is required.
Note the XhoI site has been regenerated.
Name the New Primers and Create the Product Sequence
Switch back to the Fragment tab.
Enter appropriate descriptive names for the two newly designed primers.
Enter an appropriate name for the new 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.