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Part:BBa_K801005

Designed by: Simon Heinze   Group: iGEM12_TU_Munich   (2012-10-02)


RFC25 MCS for optional addition of C-terminal Strep-Tag

This Part is a multiple cloning site and is found in TU Munich's yeast vectors pTUM100, pTUM104 (BBa_K801000, BBa_K801004). There, it lies between an RFC10 prefix (restriction sites EcoRI, XbaI) and an RFC25 suffix (restriction sites AgeI, SpeI, PstI). It has an internal NgoMIV restriction site, which is necessary for c-terminal fuison of a Strep-Tag to a CDS. Depending of the restriction enzymes used, the Streptag is either removed or added to the 3'-end of the CDS of choice.

Application of this part: For simple insertion of a CDS into the vector:

  • Use your favorite RFC10 restriction enzymes to digest both the vector and the insert.
  • Ligate and transform your E. coli strain of choice

For insertion of a CDS in front of the strep-tag, creating a "CDS-strep" fusion protein:

  • Insert must be RFC25 compatible and have an RFC25 suffix
  • Digest vector with XbaI and NgoMIV
  • Digest insert with XbaI and AgeI
  • Ligate and transform your E. coli strain of choice

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 7
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 13
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 23


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