A 5.7-kb EcoRI fragment from cosmid KE7 containing lin-14
exons 4 to 13 plus about 1.7 kb of the lin-14
3'untranslated region (3'UTR) was first cloned into the EcoRI site of pBluescript to make pB14R. An internal EcoRI site within this fragment was eliminated by partial digestion and end-filling during the cloning. The green fluorescent protein (GFP) coding sequence was cut from pPD95.02 with AgeI and SmaI and then blunted and cloned into a blunted EcoNI site of pB14R (with GFP sequences in frame with the carboxy end of lin-14
coding sequence) to make pB14RGFP. p14
GFP was made by inserting a 12.8-kb KpnI-SalI fragment from cosmid KE7 into the corresponding sites of pB14RGFP.