User GuidesSnapGene User Guide In-Fusion® CloningSimulate In-Fusion® Cloning with Multiple Inserts

Simulate In-Fusion® Cloning with Multiple Inserts

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 simulate directional in-frame fusion of the SEC13 coding sequence (CDS) and EGFP CDS into the Pichia pastoris expression vector pIB2.

Open a Vector Sequence

Import or Open the vector sequence.

In Map or Sequence view:

  • 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, vector digested with KpnI and SpeI will be used in the In-Fusion® Cloning reaction.

Start the In-Fusion® Cloning Tool

Click Actions → In-Fusion® Cloning → Insert Multiple Fragments....

When starting the In-Fusion® Cloning tool, the DNA sequence selection in the frontmost window will automatically be set as the vector region to be replaced by the inserts.

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.

The In-Fusion® Cloning tool opens showing the "Vector" tab, with the option to "Linearize with restriction enzymes" automatically selected and the appropriate fragment for replacement selected.

Set the Number of Fragments for Insertion

By default the Ligate Fragments tool starts expecting two insert fragments.

Switch to the "Fragments" tab.

Click the +/- buttons to add or remove fragments. The number of fragments is displayed in the Tab Header.

For large numbers of fragments, click the dropdown and choose "number of Fragments". Enter the number of fragments and click OK.

Define the Insert Fragments Sequences

To define the inserts use in combination of the following methods:

  1. Drag and drop files into the "Fragments" panel.
  2. Set the "Fragment" via the dropdown then click the "Source of Fragment" dropdown and choose a file, or click Browse to choose a file.
  3. Click the "Click here" links to browse and choose one or more files.

Reorder the Fragments Sequences

If fragments are in the wrong order, click the dropdown and choose "Order of Fragments".

Select one or more sequences and use the arrows to change the order.

Define the First Fragment Region

In the Fragments tab, in Map view select the Feature defining the Fragment 1 insert, or switch to Sequence view and manually select the insert region.

In the above example we plan to generate an in-frame fusion between the SEC12 CDS and the EGFP CDS.  Therefore, we switch to Sequence view and select the precise SEC13 sequence region, excluding the SEC13 stop codon.

The option to "Use as a template for PCR" will be automatically set as SnapGene assumes the need to amplify the selection by PCR.

Define the Second Fragment Region

Use the dropdown to select "Fragment 2".

In the Fragments tab, in Map view, select the Feature defining the Fragment 2 insert region, or switch to Sequence view and manually select the insert region.

In this example, we click on the EGFP CDS feature to select it as the second fragment.

Design Primers to Amplify the Insert Fragments

Switch to the Product tab and click Choose Overlapping PCR Primers....

Set the desired Target Tm for the PCR primers.

Set the desired overlap length.

If required, check one or both options to either "Regenerate" or "Remove" the partial restriction sites at the ends of the vector.

Click Choose Primers to design PCR primers for amplification of the inserts.

Confirm the Vector/Insert Fusion Points

In the Product tab, switch to Sequence view. Scroll and check the fusion boundaries between each fragment.  

In this example we confirm that the SEC13 CDS is in-frame with the EGFP CDS.

Name the New Primers and Create the Product Sequence

In the Fragments tab, use the dropdown to switch to each Fragment in turn and enter appropriate names for each new primer.

Set an appropriate name for the Product sequence, then click Assemble to create the Product sequence.

Click File → Save to save the Product sequence to an appropriate location on your computer.

View History

Switch to History view to see all steps simulated and sequences/primers created during the In-Fusion® Cloning procedure.

Order the PCR primers

See Export Primers for details on how to export and order the new primer sequences.