DNA sequence construction

A method of modifying the DNA sequence in a circular vector to give a desired circular DNA sequence which method comprises:

• (a) treating the circular vector with a restriction enzyme to obtain a linear vector having an upstream cleavage terminus and a downstream cleavage terminus;
• (b) treating the linear vector of (a) with exonuclease IH to generate 5' protruding sticky ends extending at least about 10 bases at each of the upstream and downstream termini;
• (c) mixing the resultant of (b) with an even number of single-stranded oligonucleotides, each oligonucleotide containing at least about 18 bases, wherein said oligonucleotides are comprised alternately of anti-sense and sense sequences of the desired circular DNA sequence which are missing from, and contiguous, in the desired DNA circular sequence, to the DNA sequence of the linear vector of (b), and wherein
• (i) the first oligonucleotide is an anti-sense strand and has a 5' terminus which is complementary to and overlaps at least about 8 bases of the protruding 5' sticky end of the downstream cleavage terminus;
• (ii) the last numbered oligonucleotide is a sense strand and has a 5' terminus which is complementary to and overlaps at least about 8 bases of the 5' protruding sticky end of the upstream cleavage terminus;
• (iii) each of the remaining (second through second-to-last) oligonucleotides are alternately sense and anti-sense strands each having a 5' terminus which is complementary to and overlaps the 5' terminus of the adjacent oligonucleotide, and a 3' terminus which is complementary to and overlaps the 3' terminus of the adjacent oligonucleotide;
• and
• (iv) wherein the length of the oligonucleotides is such that the regions of overlap leave single stranded portions of at least about 2 bases;
• and
• (d) treating the mixture of (c) with a DNA polymerase and a ligase, wherein the DNA sequence of the circular vector is modified.