After creating a construct for the fusion of a keratinocyte protein to the N-terminus of EGFP (pEGFP-N1) and going through several unsuccesful attempts to generate a fluorescent signal, we found by means of sequence analysis (both on DNA level and RNA level (RT-PCR, 5' and 3'RACE)) there was an additional HpaII site (CCGG) at MCS position 670 (next to Age I position).
Has anyone in anyway come about these 4 extra nucleotides or heared about their presence in pEGFP-N1?
N.B. The construct was supposed to have a "linker" of 21 nucleotides of the MCS followed by the ATG of EGFP. The extra 4 nucleotides are not in a region where digestions and/or ligations were performed.
dept. of Dermatology,
University Medical Centre Nijmegen,
-------------- next part --------------
An HTML attachment was scrubbed...