To access the Assembly Wizard, first open a sequence file. Look to the bottom of your screen and find Assembly Wizard next to Split Workspace. Click Assembly Wizard > Create New Assembly.
Select Golden Gate and press Start.
Open your backbone sequence and click the Backbone panel. You can either choose a particular selection of DNA or select specific enzyme cut sites. Change the restriction cut site by clicking on the Enzyme in blue. Press Set Fragment to set the backbone when ready.
Next, open your insert sequence and click the Insert panel. Select all or a simply a region of the insert's bases. Press Set Fragment to set the first insert.
Press the plus button on the right side of the Assembly Wizard to add another insert. Open a second insert and click the new Insert panel. Select all or just a region of your next insert. Press Set Fragment to set the second insert when ready.
If you want to insert a spacer sequence (like a "kozak"), click on one of the Insert panels and press Add Spacer After. Name the spacer “kozak” and set its bases to gccacc. Press Save to set the spacer sequence.
Rename the assembly to whatever name you desire and press Assemble at the right of the assembly wizard.
Select the folder you want to store the assembly sequences and primers. Press Create to complete the assembly.
When looking at the newly assembled plasmid, the backbone and insert fragments are colored. Open the History panel’s Lineage tab to see its lineage.
Open the Assembly tab to see an overview of the assembly, print it, and export the primers.
Note: If you change one of the bases in a primer binding site you'll notice that the primer will automatically update. That mutation will appear as a mismatch in the Assembly tab's 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.