Clone using the Gibson Assembly Wizard

  • Updated

To access the Assembly Wizard, first open a sequence file. At the bottom of your screen you will find the Assembly Wizard next to Split Workspace. Click Assembly Wizard, then select Create New Assembly.  In the options provided, select Gibson and press Start to proceed with the assembly.


Open a backbone sequence and click the Backbone slot. To linearize the backbone sequence with a restriction enzyme cut site, click the cut site, hold Shift, and click it again. Press Set Fragment to set the backbone.


Open your next insert sequence and click the Insert slot. Select all of the insert's bases. Press Set Fragment to set the first insert.


Press the + button at the right of the assembly wizard to add another insert. Open the next insert and click the new Insert slot. Again, select all the insert's bases. Press Set Fragment to set the second insert.


If you want to add in a spacer sequence like "kozak" between any 2 inserts click the press the Add Spacer After button. Name the spacer “kozak” and set its bases to gccacc. Press Save to set the spacer sequence.


Once your assembly is complete, rename the assembly to your desired construct name and press Assemble at the right of the assembly wizard.


Select the desired folder location for both the Sequence Folder and Primer Folder. Press Create to complete the assembly.


Open the newly assembled construct and click on the history panel to view the sequence lineage. Notice that the backbone and insert fragments on the assembled sequence are colored.


Open the Assembly tab to see an overview of the assembly, print it, or export the primers.


If you replace any of your sequences in your primer binding site, Benchling will automatically update the primer and will display the mutation in the Assembly's tab Mismatches Introduced table.


To enable aligning to the assembled sequence or making arbitrary edits on it, open the Assembly tab and press Finalize at the top right.

Note: The assembly wizard does not expect homology arms to be incorporated into the backbone/insert, as these are simply concatenated together.


Was this article helpful?

Have more questions? Submit a request