Required Data:

To get started, please have the sequences of your DNA fragments ready in raw or FASTA format. NEBuilder has many common vector sequences available for you, as well as a few sample insert sequences if you just want to learn. You should also have ready either the restriction sites or sequence coordinates that will define the insertion site on the vector.

Basic Usage:

1. Set preferences for the assembly, including the number of fragments to be joined and the enzyme you will use for PCR amplification. Change settings at any time and the results will be instantly updated.
2. Build your construct by adding fragments to your assembly. You can add one vector backbone and one or more inserts. Repeat as needed.
3. View primers. NEBuilder will give you a list of the required primers, their melting temperatures, recommended annealing temperatures, as well as the final sequence of your assembled construct and a summary of the design.

Additional Information:

• Click the HELP button for NEBuilder details.
• Find more about Gibson Assembly, including helpful videos, at www.NEBGibson.com.
• Gibson Assembly Cloning Kit Manual (PDF)
• Gibson Assembly Master Mix Manual (PDF)

Fragment arrangement

Notes

Required Primers