::gfp] translational fusion, a 2.3 kb SphI-AatII unc-104
fragment (including exons 510) from cosmid C52E12 was cloned into pGEM-7Zf vector and named Part II. A 3.0 kb fragment representing the 3 end of the unc-104
gene (exons 1022) was PCR-amplified from the cDNA clone yk16g10
using the primers 5'-tatgctcaacaagaacttc-3 and 5'-caactgcagtgaagcag-caattgaagatg-3 . This 3.0 kb fragment was shuttled through the pGEM-T vector (Promega) and was named Part III. Ligating the 2.3 kb SphI-AatII from Part II with the 2.7 kb AatII-PstI fragment from Part III generated a 5.0 kb fragment. This 5.0 kb SphI-PstI fragment was cloned into the SphI-PstI site in pPD95.77 and named Part II III. A 6.2kb SphI-SphI fragment from cosmid C47A5 was characterized and found to overlap a fragment of cosmid C52E12. This SphI-SphI fragment extended from 3.7 kb upstream of the first exon to the intron between exons 4 and 5 of unc-104
. The plasmid clone of UNC-104::GFP was constructed by inserting the 6.2 kb SphI-SphI fragment into Part II III in the correct orientation. --precise ends.