Opening the Gibson Assembly Modal
In order to perform in-silico Gibson assembly, start by navigating to the “Cloning” tab in the top banner. Then select the “Gibson assembly” button and the cloning modal will open.
Selecting and Linearizing the Vector
In the Gibson assembly modal box you will first have the option to select your vector. By default the current open file will be selected, if you would like to select a different file as the vector navigate to the bottom right of the modal and select “Change vector” or use the pencil icon at the top under the “Vector” tab. This will allow you to select a new file from your library or enter a sequence via copy pasting. If your selected file is a circular plasmid, you must first linearize the sequence. On the left side you will see the option to select a “Linearize by” method select the dropdown and choose between PCR, restriction digest or to use the file directly (if it is already linear). For details on the process of linearization via PCR or restriction digest see 7. Edit Options > Sequence Topology and Linearization. If you would like to perform linear ligation via Gibson it is possible to skip the vector selection and move to selecting your linear fragments by selecting “Continue without vector” at the bottom right. Once you have linearized your plasmid you can select “+ New insert” from the bottom of the modal. Note that for PCR primers will not yet have the added 5’ extensions necessary for Gibson assembly, these will be calculated and displayed at the end of the process once you have chosen the “Assemble” button.
Selecting and Linearizing the Insert(s)
After selecting “+ New insert” you will be taken to the insert selection, you can choose a file from your library under the “My sequences” tab and open it by double clicking or enter a sequence via copy and pasting in the “Enter sequence” tab. You can then linearize your insert or use it directly if it is already linear. Again the procedure of selecting a region and creating primers or selecting a linearization point and creating primers is identical to how it is described in 7. Edit Options > Sequence Topology and Linearization. Once you have finished linearizing your insert you can repeat the process for the next insert by navigating to the top of the modal and pressing the + icon next to the current tab or by navigating to the bottom right of the modal and selecting “+ New insert”. Once you are finished adding inserts, you can complete the assembly by selecting the “Assemble” button at the bottom right of the modal. Note that the assembly button will remain gray and unclickable until the fitting primers have been created for all inserts.
Completing the Gibson Assembly
After having selected “Assemble” you will see the parameter selection overview for the Gibson assembly overlaps. You have the option to select between a fixed total overlap length (by default this is set to 40bp meaning that both the vector and the insert will have a 20bp overlap for 40bps in total) or to create overlaps of variable length that optimize for a selected annealing temperature (by default this is set to 15-25bp primers with a Tm of 50C this means that the total overlap will be 30-50bps). Once you have set your parameters you can select the assemble button to automatically add the optimized overlaps to your primers and simulate the final assembly.
After selecting assemble you will see a preview of the final construct. You can adjust the overlap regions and re-assemble if desired. Additionally, you can now navigate to the “Inserts” and “Vector” tabs at the top in order to inspect the generated primers, the overlaps on the primers are displayed in red. Once you are finished use the “Save assembled product” button at the bottom right of the modal to save the final product to your library.
