The synthetic sGFP(S65T) gene was generated from six fragments of approximately 120 bp each, produced on a Milligen 8750 synthesizer, After elution with 30% ammonium hydroxide, the oligonucleotides were deblocked at 55^oC for 12 h, precipitated with n-butanol and resuspended in H₂0. 15-25-mer oligonucleotides complementary to the ends were used to amplify the long oligonucleotides by PCR. Typically, PCR was carried out using 35 cycles with 55^oC annealing temperature and 0.2 min extension time. The products were gel purified, phenol extracted, and used in a subsequent overlap PCR to generate longer fragments consisting of two adjacent small fragments, using a strategy for assembly that relied on the ability of the restriction enzymes BsaI and BbsI to cleave outside their recognition sequence. Long oligonucleotides were synthesized which contained portions of the coding sequence of GFP embedded in flanking sequences encoding EcoRI and BsaI sites at one end, and BamHI and BbsI sites at the other end, in the configuration EcoRI-BsaI-GFP-BbsI-BamHI. The restriction site ends generated by the BsaI and BbsI sites were designed to yield compatible ends between adjacent GFP fragments, each of which was unique and non-selfcomplementary. The crude synthetic DNA segments were amplified by PCR, inserted between EcoRI and BamHI in pUC9, and sequenced. Subsequently, the intact coding sequence was assembled in a six fragment ligation, using insert fragments prepared with BsaI and BbsI, Two of six plasmids resulting from the ligation bore an insert of correct size, and one contained the desired full length sequence. Mutation of Ser65 to Thr was accomplished by PCR, using a primer that overlapped a unique BssSI site in the synthetic GFP (Haas J, Park E-C, Seed B: Curr Biol 1996, 6: 315-324).