2. Gibson assembly is novel method for the easy assembly of multiple linear DNA fragments. Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule.

3. Forming 3’ overhangs Annealing complementary termini Gap-filling Nick-sealing

5. T5 Exonuclease -creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other. Phusion DNA Polymerase -incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments. Taq DNA Ligase -covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.

6. 1. T5 exonuclease excises nucleotides from the 5 ′ end of dsDNA molecules, exposing 3 ′ overhangs. (Similar reactions will occur at all DNA termini, but for the sake of simplicity we show one assembly junction only). 2. Complementary 3 ′ overhangs anneal. 3. T5 exonuclease (unstable at 50°C) denatures, exposing free 3 ′ overhangs to Phusion DNA polymerase.

7. 4. Phusion DNA polymerase fills the 3 ′ overhang. 5. The DNA polymerase releases the DNA once the overhangs are filled, allowing Taq DNA ligase to seals nicks in the phosphodiester backbone (i.e. catalyse the formation of phosphodiester bonds between 5 ′ phosphate and 3 ′ hydroxyl groups).

9. No need for specific restriction sites. Join almost any 2 fragments regardless of sequence. No scar between joined fragments. Fewer steps. One tube reaction. Can combine many DNA fragments at once.

10. Cost and delivery time of custom primers. Rearrangements are possible between regions of homology close to the termini of substrate sequences (at low frequency). Secondary structure can reduce assembly efficiency.

11. https://www.youtube.com/watch?v=uX9LWk1Hzv4