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

Arno Pol vetpol at telebyte.nl
Tue Jul 18 04:03:27 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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