Simulate Gateway® Cloning with One Insert
How do I simulate Gateway® cloning in SnapGene?
SnapGene can simulate BP- or LR-type reactions, or a combination of both, with 1 to 4 ordered inserts.
SnapGene provides a library of commonly used Gateway donor and destination vectors.
Gateway® is a registered trademark of ThermoFisher Scientific. See Gateway Recombination Cloning Technology to learn more about Gateway® Cloning.
See our Gateway Cloning video for more about how to simulate Gateway cloning in SnapGene.
In this lesson we do the following:
- Define an insert sequence for PCR amplification and subsequent cloning via BP + LR reactions.
- Have SnapGene design appropriate PCR primers to add attB sites to the insert.
- Simulate a BP reaction between the insert PCR product and the donor vector pDONR221.
- Simulate an LR reaction between the pDONR221:insert plasmid and the destination vector pDEST17.
In this lesson we will fuse a coding sequence (CDS) in-frame with the N-terminal HIS-tag encoded by the pDest17 expression vector.
Define the Insert Fragment
An open DNA file will automatically be preselected as the insert for Gateway cloning.
Open the DNA file containing the insert sequence. Select the region comprising the desired insert. Select a feature in Map view, or switch to Sequence view to select a specific nucleotide range.
Start the Gateway Cloning Tool
Click Actions → Gateway® Cloning → and choose the reaction, or combination or reactions, to simulate.
In this lesson we will simulate BP + LR reactions in one operation.
The "attB Fragment" tab is displayed and the selected region of the foremost open sequence file is automatically selected as the insert.
If required, click the "Source of attB insert" dropdown menu to choose an alternative insert sequence.
Set the Donor Vector
Switch to the "Donor Vector" tab and choose the donor vector from the list of "Standard Donor Vectors".
If you wish to use a "custom" donor vector then select the option "Custom Donor Vector" and use the dropdown menu choose an appropriate donor vector from your sequence files.
Set the Destination Vector
Select the "Destination Vector" tab and choose the destination vector from the list of "Standard Destination Vectors".
If you wish to use a "custom" destination vector then select the option "Custom Destination Vector" and use the dropdown menu choose an appropriate destination vector from your sequence files.
Create attB PCR Primers
Select the "Expression Clone" tab and click Choose attB PCR Primers" to have SnapGene design appropriate PCR primers to amplify the insert.
- Set whether the insert is recombined in the forward (attB1 → Insert → attB2) or reverse (attB2 → Insert → attB1) orientation.
- Set the desired target melting temperature (Tm) for the new PCR primers.
- Check the option to "use attB1 and attB2 adapter primers" if you wish to design and use additional attB1 and attB2 adapter primers†.
Click Choose Primers to design appropriate primers for the insert.
† ThermoFisher recommend using adapter primers to produce attB-PCR products if your attB PCR primers are longer than 70 bases. See the Gateway manual for details about when and how to use attB adapter primers.
View the Expression Clone
Switch to the "Expression Clone" tab. The predicted "Expression Clone" will be shown in Map view, the binding sites of the new attB primers will be displayed, the insert will be colored red.
Check Insert/Vector Fusion Boundaries in the Expression Clone
In the "Expression Clone" tab, in Map view, click and select the insert.
Switch to Sequence view and scroll to confirm the insert in positioned correctly in the vector.
If required, scroll down and check the fusion boundary at the end of the insert.
In the above example, the pDest17 vector start codon and HIS-tag of the expression vector are confirmed to be in-frame with the insert CDS.
Name the New Primers
Switch to the "attB Insert" tab to view the new primers.
Give the primers appropriate descriptive names (optional).
Confirm the primers are shorter than 70 nucleotides. If longer than 70 nucleotides then consider using adapter primers (see above).
Simulate Gateway Cloning
Switch to the "Entry Clone" tab, enter an appropriate name for the intermediate "Entry" plasmid.
Switch to the "Expression clone" tab and enter an appropriate name for the new "Expression" plasmid.
Click Clone to create new "Entry" and "Expression" plasmid sequences.
Click File → Save to save each new sequence to an appropriate location on your computer.
View the Expression Vector History
View the Expression clone sequence and switch to History view to see new primers, cloning steps and and all intermediate sequences generated during the Gateway cloning simulation.
Order Primers to Start Gateway Cloning
Export your attB primer sequences as text for ordering from an oligonucleotide synthesis service provider. See Export Primers for more information on how to export primers.