Simulate Gateway® Cloning

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.

For your convenience, SnapGene provides a library of commonly used Gateway donor and destination plasmid 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.

An open DNA file will automatically be preselected as the insert for Gateway cloning.

In this lesson we will do the following:

1. Define an insert sequence for PCR amplification and subsequent cloning via BP & LR reactions.

2. Have SnapGene design appropriate PCR primers to add attB sites to the insert.

3. Perform a BP reaction between the insert PCR product and the donor vector pDONR221.

4. Perform an LR reaction between the pDONR221:insert plasmid and the destination vector pDEST17.

In this lesson we will "clone" the CDS region encoding the XynB mature enzyme (without signal peptide), in-frame with the N-terminal HIS-tag encoded by the pDest17 expression vector.

Define the Insert and Start the Gateway Cloning Tool

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.

Click Actions → Gateway® Cloning → and choose the reaction, or combination or reactions, to simulate.  

Initally the "attB Insert" 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 alternate insert sequence.

Click the "Polymerase for making attB Insert" dropdown menu to set the polymerase "type" used for PCR amplification of the insert.

Set the Donor Vector

Select 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.

1. Set whether the insert is recombined in the forward (attB1 → Insert → attB2) or reverse (attB2 → Insert → attB1) orientation.

2. Set the desired target  melting temperature (Tm) for the new attB PCR primers.

3. Check the option to "use attB1 and attB2 adapter primers" if you wish to design and use additional attB1 and attB2 adapter primers.

ThermoFisher recommend using adapter primers to produce attB-PCR products if your attB PCR primers are greater than 70 bp. See the Gateway manual for details about when and how to use attB adapter primers.

Click Choose Primers to design appropriate primers for the insert.

View the Expected Expression Clone, attB PCR Primers, and Insert/Vector Fusion Boundaries

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.

Switch to the "attB Insert" tab to view the new primer sequences. Use the fields provided to edit the default primer names (optional).

The insert-specific portion of each primer is colored black. The attB portion of each primers is colored red.

Confirm the primers are shorter than 70 nucleotides. If longer than 70 nucleotides then consider using adapter primers.

Switch to the "Expression Clone" tab, in Map view, select the insert feature.

Switch to Sequence view to confirm the insert CDS is in-frame with the start codon and HIS-tag of the pDest17 expression vector.

If required, scroll down and check the fusion boundary at the end of the insert.

Simulate Gateway Cloning

Once sequence checks are complete, 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 the predicted recombinant "Entry" and "Expression" plasmid sequences.

New unsaved files will be created.

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 then switch to History view to see all  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.