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Golden Gate Cloning of Multiple Fragments into a Vector with no Type IIS sites

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How do simulate Golden Gate cloning of multiple fragments into a Golden Gate vector that does not have suitable Type IIS enzyme sites?

In this lesson SnapGene will design primers for addition of appropriate Type IIS enzyme sites to all input sequences (vector and inserts) to facilitate Golden Gate Assembly.

Open or Import a  Vector Sequence

Open or import the vector sequence.

Choose an Appropriate Golden Gate enzyme for Cloning

Click Enzymes → Choose Enzymes, set to "Choose from" the "Golden Gate Enzymes" set.

Uncheck the option to "Hide Noncutters" to show all Type IIS enzymes that do not cut the vector.=

In this example PaqCI is determined not to cut the vector and will be used for the Golden Gate procedure.

Be sure to also confirm the selected Type IIS enzyme does not cut within any of the intended insert sequences.

Start the Golden Gate Tool

Click Actions → Golden Gate Assembly → Insert Multiple Fragments... to start the Golden Gate tool.

Set the Golden Gate Enzyme

The open file will automatically be set as the vector. If required, use the "Vector:" dropdown menu to choose the vector sequence.

Use the "Golden Gate Enzyme:" dropdown menu to set the enzyme to use for the Golden Gate Assembly. Choose Settings... from the dropdown if the enzyme is not in the default list, see Set the Enzymes for Golden Gate Assembly.

In this example, the vector does not have PaqCI sites, so the option to "Run PCR and digest with PaqCI" is automatically set as SnapGene assumes the need to amplify the vector by PCR. SnapGene will design primers to add appropriately positioned PaqCI sites to the vector PCR product.

Select the Region for Replacement in the Vector

In the Vector tab, use Map view to select a feature for replacement, or switch to Sequence view to select a specific sequence region for replacement.

Define the Insert Fragment Sequences

Switch to the Fragments tab.

To set the insert sequences use any 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 from the list of "Open" or "Recent" files, or click Browse to choose a file.
  3. Click the "Click here" links to browse and choose one or more files.
  4. Use the + and "-" buttons to add or remove fragments.

Reorder the Fragments Sequences

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

Select one or more sequences in the list and use the "Move:" controls to reorder, then click OK.

Define the First Fragment Region

In the Fragments tab, if not selected, use the dropdown to select "Fragment 1".

In Map view select a feature to define the fragment 1 insert, or switch to Sequence view and manually select the precise insert region.

In this example we plan to generate an in-frame fusion between the xynB CDS and mCherry CDS.  Therefore, we switch to Sequence view and select the xynB CDS sequence region, excluding the xynB "TAA" stop codon.

The option to "Run PCR and digest with PaqCI" will be automatically set as SnapGene assumes the need to amplify the selection by PCR. SnapGene will design primers to add appropriately positioned PaqCI sites to the ends of each insert fragment.

Define All Other Fragment Regions

In the Fragments tab, use the dropdown, or the left/right arrows, to select each "Fragment" in turn.

In Map view, select the Feature defining the Fragment 2 insert region, or switch to Sequence view and manually select a precise insert region.

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

Design Primers to Amplify the Vector and Inserts

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

  1. Set the desired Target Tm for all PCR primers.
  2. Set the number of nucleotides to insert upstream of the Type IIS enzyme site that will be added to each primer†.

Click Choose Primers to design PCR primers for amplification of all fragments that require addition of enzyme sites.

† SnapGene allows you to add 2 to 9 nucleotides 5' to the type IIS site. These additions are to ensure efficient digestion by the selected enzyme, see this useful NEB page for advice on enzyme cutting efficiency for sites close to fragment ends.

View the Predicted Assembly

In the Product tab, information about the predicted Golden Gate product is displayed.

  1. The predicted product will be shown in Map view.
  2. A list of all fragments will be shown in the right hand panel, showing the overhang for each fragment ligation.
  3. A schematic of the product is displayed, showing the overhangs used for each fragment.
  4. SnapGene reports the predicted assembly fidelity in the bottom right panel. If SnapGene detects an issue with assembly, an error will be reported in this panel.

Confirm the Vector/Insert Fusion Points

In the Product tab, switch to the Sequence view to check all fusion boundaries and confirm the desired assembly is simulated.

In this example, we confirm the insert CDS is in-frame with the vector-based start codon and N-terminal tags.

Adjust Overhangs (Optional)

Click Adjust Overhangs... to change overhangs.

SnapGene is often able to choose from a range of overhangs that can be used for a scar-less fusion. SnapGene will automatically choose the overhang with the highest predicted fidelity.

Use the dropdown to look at each junction and the corresponding available overhang options.

If required, choose an alternative overhang from the list of possible overhangs, then click OK to design new primers that will specify the selected overhang.

The Adjust Overhangs panel also allows confirmation of correct in-frame fusion of adjacent CDS reading frames.

Name the New Primers and Create the Product Sequence

  1. Switch back to the Fragments tab
  2. Click the dropdown and switch to each insert Fragment in turn.
  3. Edit the primer name fields and enter appropriate names for each newly designed primer.
  1. Switch to the Vector tab.
  2. If required, edit and add appropriate names for each newly designed primer.
  3. Set an appropriate name for the Product sequence.

Click Assemble to create the Product sequence.

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

When viewing the Product file, click the "Show colors" button in the side toolbar to show History colors.

View History

Switch to the product sequence History view to see all simulated steps and all sequences/primers created and used during the Golden Gate Assembly procedure.

Click on the "Golden Gate Assembly" label to see History colors.

Order the PCR primers

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

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