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Golden Gate Cloning into a Vector Cut with non-Type IIS Enzymes

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How do I do Golden Gate assembly using a vector cut with regular type II restriction enzymes?

SnapGene allows use of two non-type IIS enzymes as the vector input for a Golden Gate reaction with the following caveats:

  • The overhang length an direction for each enzyme matches those for the chosen Type IIS enzyme
  • The selected Type IIS enzyme does not cut within the vector sequence

For example, the Type IIS enzyme BsaI generates a 5'- four nucleotide overhang. Enzymes such as BamHI, EcoRI, HindIII, NotI, XbaI, XmaI (and many others) can create overhangs compatible with 5'- overhangs generated by digestion with BsaI.

Open a Plasmid Vector with Suitable Sites for Cloning

Open a suitable vector sequence.

In this example we will used the vector-based XbaI and BamHI sites. These enzymes create 4 nucleotide 5'-overhangs compatible with overhangs generated by BsaI.

Perform Golden Gate Assembly

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

Define the Vector Region for Replacement

If a DNA file is open when you run this tool it will be automatically selected as the vector. If required, use the "Vector:" dropdown menu to choose an alternative vector file.

  1. Select the Golden Gate (Type IIS Enzyme) to use.
  2. Select the option to "Digest with other Enzymes".
  3. Enter enzyme names in the fields provided, or use the dropdown menus to choose a pair of appropriate enzymes with unique overhangs compatible with your selected Golden Gate enzyme.
  4. Ensure the correct fragment for replacement by the inserts is selected.

The selected enzymes will be displayed in Map and Sequence view, and the "Region to replace" will be selected.

Define the Insert Fragment

Switch to the Fragment tab.

To define the insert fragment use one of the following methods:

  1. 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, or
  2. Click the "Click here" link to browse and choose a files, or
  3. Drag and drop a file into the "Fragment" panel.

If your Insert fragment sequence already has a pair of appropriate flanking Type IIS sites then these will be automatically detected and selected, and the option to "Digest with ..." will be set to match.

If your insert fragment sequence does not have Type IIS sites then the option to "Run PCR and then digest with ..." will be selected.

Design Primers to Add Type IIS sites to the Insert Fragment via PCR

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 the fragment.

† 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.

Name the New Primers

Switch to the Fragment tab, and give the primers appropriate names.

View the Ligation Fidelity Details

Click the Details... link to view a matrix showing predicted overhang interaction (mis-pairing) for all overhangs present in the Golden Gate Reaction.

SnapGene will warn that the overhangs are palindromic.  Palindromic ends will reduce the predicted "Assembly fidelity" as they allow insert-insert ligation and vector-vector ligation, and consequently, may result in a higher frequency of incorrect assembly.

In this example the palindromic overhangs of the site result in a predicted ligation fidelity of 25%. In this case, the lower fidelity cannot be avoided due to the use of the vector-based XbaI and BamHI sites.

Validate and Create the Product

Switch to the Product tab, switch to Sequence view and confirm the vector/insert boundaries are correct.

Enter an appropriate name for the new product, then click Assemble to create the new product file.

View the Product History

View the product file.

Switch to History view to see all steps simulated and all sequences/primers created 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 primers.

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