Ensure you are using only one Type IIS restriction enzyme, not two distinct ones (one of BsaI will work; one of BsaI and one of BsmBI will not work)


Then, check that your insertion region is correct:

1. Find out where the cut-sites are located on the plasmid (using the restriction digest tool) and highlight in between the two cutsites. Take note of the start and end locations. 

2. Return to your CRISPR analysis, select your good guides and click assemble. Choose your plasmid

3. When choosing your "Insertion Region," insert the start and end values you identified in step 1, taking into account and adjusting for overhangs correctly. (If you directly clicked on the BbsI markers, you would have identified that the BbsI markers were located at 4462 and 4484. However, these do NOT take into account the length of the 4bp 5' overhang and that you want the insertion region to end right before the beginning of the second cut site).

Now your assembly should work properly

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