Circular Assembly Amplification

Design plus  and  minus strand oligonucleotides  in such a way that, upon annealing, complementary oligos would overlap as a circular structure, leaving no gap to fill. 

Design one more guiding oligo (24th minus strand)  to join 5’ and 3’ ends of the  target sequence.

The length of each oligo is approximately 40 to 50 base pairs, and the overlap of each oligo should be designed to have a melting temperature of 60ºC by the nearestneighbor method. 

ATG ATC GTT CTG TTC GTT GAC TTC GAC TAC TTC TAC GCT CAG ~ GTT GAA GAA GTT CTG AAC CCG TCT CTG AAA GGT AAA CCG GTT G