Stage 2 - Performing the Assembly
With your backbone vector and six sequences selected, on the Tool bar click Cloning → Golden Gate... to start the Golden Gate tool. This will open the Golden Gate Window.
If not already selected, set the Enzyme: to BsaI.
We wish to use the pGoldengate-SE7 vector as our backbone. By default, Geneious Prime should set the circular sequence as the left most sequence, and set Backbone: to Use Leftmost. If the pGoldenGate-SE7 sequence is not leftmost, then select and drag it to the leftmost position. Alternately, you can use the Backbone: dropdown menu to specify that pGoldengate-SE7 should be the backbone You can also use the Choose... button to navigate to this tutorial folder and select the pGoldengate-SE7 vector sequence as the backbone.
Each sequence is shown as a boxed "Tag" that represents a Golden Gate Part with BsaI-generated overhangs.
If your Parts are in a different order to that shown in the figure above, drag and drop the Tags to rearrange them to the correct order.
If the BsaI sites in the Parts are preexisting, such as the two in the backbone, the Tag will be labelled with Cut by BsaI, or Enzyme. Parts that require design of a primer pair will be labelled PCR product or Primer. Each Tag also provides information on the range of the original sequence that will be incorporated into the final Golden Gate construct.
If preexisting type IIS sites on your fragments are incompatible with sites on adjacent fragments then Geneious will highlight the Tag red and the offending overhangs will also be red. For example, if you switch the order of fragments GFP1 and GFP2, you will see that the Backbone and GFP2 fragments turn red and are labelled Mismatched. This is because the GFP2 sequence has preexisting BsaI sites that are not compatible with the backbone BsaI sites.
Drag and drop to place the GFP1 and GFP2 Tags back in the correct order.
Checking the correct primer_bind boundaries are used
Each part has a dropdown menu accessible via an inverted triangle. This menu can be used to check, and if required, change whether existing primer_bind annotations are used as boundaries. The GFP1 sequence provided for this tutorial contains a primer_bind annotation called "A primer". Unless told otherwise, Geneious will assume this annotation defines a Part boundary. In this case we want Geneious to ignore this annotation. To do this, click on the triangle on the GFP1 Part and change the "forward" boundary from A primer to Design at 5' end.
Other Options
The Parts drop-down menu also provides an option to Reverse Complement Parts that are in the wrong orientation. You also have the option to Reset reaction if you have previously specified non-default boundary options for the Part.
All of the Parts in this exercise, with the exception of the Backbone and GFP2, will require primer design and PCR in order to generate DNA suitable for the Golden Gate reaction. Therefore, make sure the option to Save used Primers is checked. This will provide you with a file for each primer sequence designed by the tool. These sequences can then be submitted to an oligonucleotide synthesis company.
Uncheck the Save intermediate products option as we do not need to see intermediate products.
Finally, we will save our results to the same folder so make sure the option to Save in sub-folder is unchecked.
For more information on the various options available in the Golden Gate window, you can click on the Help button in the bottom left corner of the window.
Completing the Golden Gate Assembly
Press OK to run Golden Gate.
The Geneious Golden Gate tool will then complete the analysis and save a circular construct that represents your assembled sequences, plus the primer sequences required to create the Parts.
Go to Stage 3: Checking the Assembly