Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene

The construction of the H1V-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (>100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat</it> gene was assembled in three successive cycles adjacent to the lac</it> promoter in the pEMBL-derivative, pKH125. The 15 kD tat</it> protein was produced from this synthetic gene in E. coli</it> upon IPTG induction. However, it was necessary to tightly control the expression of tat</it> by including the lac</it> I gene directly within the tat expression vector.