extra HpaII site in pEGFP-N1's MCS???

arno pol vetpol at pop.telebyte.nl
Tue Jul 18 02:29:15 EST 2000


Dear fluorpro's,

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.

greetings,

Arno Pol
dept. of Dermatology,
University Medical Centre Nijmegen,
the Netherlands.
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