Cloning → Digest into Fragments. Ensure only NcoI is selected and click OK.We want to create a Decorin(DCN)-eGFP fusion gene using Gibson Assembly.
First, we open the Vector which already contains the eGFP. We want to insert DCN just before the start codon of the GFP, so highlight the GFP CDS annotation and zoom in to the 5' end. Turn Translation on if it's not already activated (on the right hand side of the Sequence Viewer click on 'Display' → 'Translation'). The Gibson assembly works by joining sequence extremities together, so first we have to provide this 'extremity' by linearizing our vector just before the ATG codon using either PCR or restriction digestion. As there is already a unique NcoI restriction site we can conveniently use this to digest our vector. To do this go to Cloning → Digest into Fragments. Ensure only NcoI is selected and click OK.
The resulting sequence now bears two 5' overhangs which will be digested by T5 exonuclease during the Gibson Assembly. Removal of this overhang will keep our protein in-frame, but will remove the start codon (ATG). In order to keep the ATG and not truncate the CDS in any way we can add it again to the 5' end of the vector. Turn on Allow Editing and add the missing 'ATG' bases manually as shown in the figure below.
add primer icon.
We're finished with the vector, so let's head over to DCN. We want only the CDS to be inserted before GFP, without the stop-codon. Translation should still be turned on, select the CDS bases except for the last TAA (for example by clicking on the CDS annotation, then holding Shift+click just before the 'TAA'). We're going to extract this without the use of primers, as they will be generated later by Geneious during the Gibson operation. Click on Extract in the Sequence Viewer and chose an appropriate name, such as "DCN CDS".
Now select both the extracted DCN CDS and the digested vector sequences. Bring up the Gibson Assembly options (Cloning → Gibson Assembly...). In the Backbone dropdown select the digested vector if it is not already selected. After the vector is selected as the backbone, the tag representing this sequence in the Construct Layout panel should turn green, and the insert tag should be grey, as in the screenshot below. Click the tags to see a preview of the sequences that will be used in the assembly.
Note that in Geneious 8.1 and above, the inserts and vector do not have to be pre-selected before opening the Gibson Assembly tool. Instead, they can be selected from within the setup options by clicking the Choose button.
Check that the Exonuclease is set on 5' Exonuclease. If you are using a related method that uses a different exonuclease (such as SLIC or In-Fusion cloning), you can choose this here.
Now click Primer Options to check the primer settings. The Min Overlap Length is set to a recommended value of 18bp. This is the length of the annealing bases, the complementary sequence part that two neighbouring sequences will have in common after the operation. When ligating two inserts, each will have half of this length as primer extension. In our case we're ligating one sequence into a vector and because primers are only created for insert sequences the insert primer will have the full 18bp as extension. Min Overlap Tm (48°C) is the melting temperature of the complementary sequences. The Tm settings are used for the extension as well as the primer binding site (both get calculated independently). Most labs are using Phusion Polymerase, for which the recommended Tm gets calculated after a formula invented by Breslauer et al., so select this option under Formula. Confirm this dialog by clicking OK, then click OK in the main Gibson Assembly options to run the analysis.
Once the assembly has run, a new folder containing the Gibson Report, ligated sequence product, and generated primers will appear in the Document Table. Open the Gibson Report. In the Hints section you will see a note that the 5' overhanging bases that were created by the NcoI digestion have been removed from the sequence, and the bases we annotated as "Gibson Primer Extension" have added to the primer.
The Generated Primer table lists the primers required for this reaction, and the primers listed here also appear as separate files in the Document Table. Primers are designed to bind to the Insert sequence, with the extension homologous to the flanking vector sequence. In the table you will see that the forward primer (
) contains the extension for the left flanking sequence, and the reverse primer (
) has an extension for the right flanking sequence. If you look on the reverse primer extension in green you will notice the 'cat' bases (highlighted in red in the screenshot) - these are the reverse complement of the bases for the start codon that we added as a Gibson Primer Extension. The total length of the extension has been extended by the length of the added start codon, so that the extension is 21 bp long in total, but the complementary bases between the insert and the vector are still 18 bp long.
To check the results we can click on the link to the Product in the Gibson Report. The DCN CDS has been inserted before the GFP CDS and is flanked by both primers. When you hover over the primer annotation you will see the characteristics of the primer, the binding sequence and the extension sequence.
Scrolling in a bit further to the transition of the insert to the GFP we will notice the inserted ATG which is annotated as a manually inserted sequence. The translation shows this as a methionine and the GFP CDS is perfectly in frame.
You may want to check whether the melting temperatures for the primers are ok before you order them.
Congratulations, you successfully simulated a Gibson Assembly within Geneious.