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How to Assemble Complex Synthetic Genes in 5 Steps

Are you tired of secondary structures limiting the synthesis of your gene? Researchers trying to synthesise long (>2.5 kb) or complex GC/AT rich genes often encounter problems during synthesis due to the formation of secondary structures within the DNA.

With these 5 steps, it no longer needs to be an issue.

1. Synthesise your long or complex GC/AT rich gene as a series of smaller synthetic gene fragments, split at specific regions to avoid the formation of secondary structures, but including the 20 bp overlaps required for multiple fragment cloning using Takara’s Next-Gen In-Fusion HD® Cloning Plus System.

2. Linearise the vector that you want to assemble or clone the synthetic fragments into at the desired locus via inverse PCR to be completely restriction site independent, or via restriction enzyme digestion.

3. Incubate the various synthetic gene fragments and the purified linearised vector with the In-Fusion HD enzyme for 15 minutes at 50°C. The In-Fusion HD enzyme will then create ssDNA regions at the ends of each insert/vector and allow the inserts and vector to spontaneously anneal at the exposed homologous 20 bp overlaps (see Figure 1 below). This allows the various fragments to be effectively assembled and cloned as a single fragment.

synthetic-biology-cloning-gene-fragmentsFigure 1: Cloning multiple fragments in a single reaction.

4. Transform the In-Fusion HD multiple fragment cloning reaction into our Stellar competent cells where any gaps will be repaired in vivo resulting in positive clones containing the recombinant vector with the various assembled inserts.

5. Screen positive clones for the presence of your assembled gene fragments –  you should only need to screen 2-3 clones per construct before obtaining your positive clones due to the high cloning efficiency of In-Fusion HD (> 90% for multiple fragment cloning).


Malathi Raman
, Takara Bio’s European Product Manager, will be presenting at the Synthetic Biology and Gene Editing Strand of the 4Bio Summit. Malathi’s presentation will discuss accelerating synthetic gene and protein production.

Find out more about Takara Bio’s presentation.

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