Designing Clones Using Benchling

First, go to [1] to create an account. The following protocol is a tutorial for designing clones using the Gibson method:

1. Click the "Create+" tab at the top righthand side of the page.
2. Scroll down to "Create sequence".
3. Select "Design assembly".
4. Select "Gibson" and hit "start".
5. Towards the bottom lefthand of the page there are tabs to set your 'backbone' plasmid and 'insert' the desired region of your gene of interest.
6. Both the backbone and the gene can be uploaded from the site if you go back to the "Create+" tab, scroll down to "Create sequence" and select "Import from File/DB", then you have the option to "Search external databases". From here you can type in the gene or plasmid you are looking for and choose the proper 'genome' for designing primers for your clone and you can also specify if you want to include upstream/downstream regions of the coding sequence. Make sure to save this information to a location that is easy to find later.
7. Select the regions you want to include just by clicking and dragging that particular portion of the sequence (for the plasmid, this would be the entire sequence).
8. Once you have selected a region, the option to "set fragment" will be available. Click that. Then do the same with the other ("insert"/"backbone").
9. When both of the fragments are set, you will have the option to "assemble", it is a good idea to create a name for you assembly (default is 'untitled assembly', just delete and type to change) prior to selecting "assemble". If you forget to do this you can click the "re-open" option under the 'assembly' tab at the top righthand side of the page, but I think this creates a duplicate assembly.
10. When you are happy with everything, you can choose the "finalize" option (located under the 'assembly' tab).
11. Benchling will automatically design primers for your assembly (they will be towards the beginning and end of the regions of your gene and to those connecting portions of the plasmid around the insert), you can find the forward and reverse primer sequences under the 'assembly' tab as well. They will show up when you click on them. From here you can just copy and paste the primer sequences into the IDT website to order.
12. There is an option to create a PDF document to save your assembly to your computer if you choose to do so, otherwise it will be available on the website.