It is not clear from your question how much is the separation between each genes.

If you have appropriate overlap (~20bp) between pUC19 backbone and gene A, gene A and gene B, and gene B and gene C and finally gene C and pUC19 backbone, you should be able to assemble them all at once. using Gibson assembly kit.

Hi Ramesh,

To clarify, gene A is GOI(gene of interest), and gene B and C are respective left, and right homology arms.

It isn't challenging to clone gene A into pUC19 by gibson assembly, but I found it confusing to clone gene B and C, which should go in at different sites (B to the left of GOI, and C to the right of GOI). If I am right, this requires multiple gibson assembly reactions? If I try to assemble them all at once, then it would require assembly of four segments (pUC19 backbone, gene A, gene B, then gene C) by gibson assembly, which makes me worried about low efficiency of the reaction due to many number of fragments to assemble..?

Gibson assembly is capable of assembling 4 DNA pieces.

Here's another trick.

Considering B, A, C have some overlap, when you got the genes, mix them all in equal molar ratio, and PCR amplify for 8-10 cycles to get B-A-C. Then amplify with primers that correspond to 5' end of B and 3' end of C, for may be 25 cycles. Gel purify to get pure B-A-C.

Gibson assemble with pUC19 backbone.

That sounds like a great idea!

Except I didn't tell you, B-A-C would be 3.5kb in size, which I assume would make cloning more challenging. I am trying to get around by cloning in the exogenous genes by splitting into multiple assemblies so that the insert sizes are smaller = easier cloning.

Thank you for your advice and for your time!