The gfp gene, along with the translation initiation region (TIR) can be excised with one of eight restriction enzymes (HindIII, PstI, SalI, XbaI, BamHI, SmaI, SacI or EcoRI). A green fluorescent protein (GFP) cloning cassette vector designed specifically for use in the construction of prokaryotic transcriptional fusion. The gfp allele in pGreenTIR contains both the F64L and S65T mutations that increase protein solubility and cause a 'red-shift' in the excitation maximum from 395 nm to 490 nm. The vector was constructed by cloning a mutant GFP gene into the EcoRI site of pUC1813. The resulting construct was mutagenized via PCR to 1) restore the 5' end of the gene to wild-type, 2) incorporate an upstream translational enhancer and 3) change the Shine-Delgarno region (and the surrounding bp) to consensus. |
