Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids....

The construction of the HIV-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 (greater than 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 gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.