How do I Simulate Gibson Assembly in SnapGene?
To watch a video on Simulating Gibson Assembly® in SnapGene see this link.
Open the Vector Sequence
Import or open a sequence file for the plasmid vector that will be used for Gibson Assembly®.
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 the pIB2 plasmid linearized with SmaI will be used as vector for Gibson Assembly.
Start the Gibson Assembly Tool
Click Actions → Gibson Assembly® → Insert Fragment....
When starting the Gibson Assembly tool, the site or DNA sequence selection in the foremost window will be automatically set as the vector region for insertion/replacement by Gibson Assembly.
If a vector sequence is not open when you start the Gibson Assembly tool then you can choose and define the vector in the Vector tab.
In this example, the Gibson Assembly tool opens showing the "Vector" tab, with the option to "Linearize with restriction enzymes" automatically selected, and the vector SmaI site selected.
If required, set the vector orientation using the "Orientation of Vector" buttons.
Specify the Insert Fragment Source Sequence
There are a number of ways to specify the fragment source sequence, you can use any one of the following methods:
Switch to the Fragment tab.
- 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 a 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, the EGFP CDS feature has been selected as the insert. The yellow box indicates that the insert requires PCR primers to generate overlaps suitable for Gibson assembly.
Select the Product tab and click Choose Overlapping PCR Primers....
Set the target Tm for the PCR primers.
Set the desired overlap length and Tm for Gibson Assembly.
If required, choose to regenerate one or both halves of the restriction site via inclusion in the PCR primers.
Click Choose Primers to design primers to allow PCR amplification of the insert and enable Gibson Assembly with the vector.
Name the Primers
Switch to the Fragment tab and enter appropriate names for the newly designed primers.
Create the Gibson Assembly Product
Enter a name for the Gibson Assembly product and click Assemble.
A new product file will be created. Click Save to save the product to an appropriate location on your computer.
View the Product History
Show History view to see a summary of the PCR and Gibson Assembly steps. To illustrate in History view where the insertions occurred, click the Gibson Assembly operation name.
Order the PCR primers
See Export Primers for details on how to export and order the new primer sequences.