r seq id no 7 with restriction sites sac i and bam hi at the 5 u2032 and 3 u2032 ends respectively paat was amplified from the a niger genome using the primers aat f seq id no 8 and aat r seq id no 9 with restriction sites eco ri and bam hi at the 5 u2032 and 3 u2032 ends respectively gfp seq id no 10 was synthesized with coden optimization and contained bam hi and pst i restriction sites at the 5 u2032 and 3 u2032 ends respectively trp terminator ttrp was pcr amplified with primers ttrp f seq id no 11 and ttrp r seq id no 12 using pan7 1 as a template and restriction sites pst i and hin diii was added to the 5 u2032 and 3 u2032 ends respectively ttrp was digested with hin diii and pst i gfp was digested with bam hi and pst i and the two sequence were ligated to puc18 digested with the same enzyme and pgt was obtained gfp ttrp was amplified with the primers gfp f1 seq id no 13 and ttrp r using pgt as a template and reversely connected to pmd19 t vector takara tokyo japan to generate pmd gfp ttrp pgas and pmd gfp ttrp were digested.