Coding

Part:BBa_K1607011:Design

Designed by: Jiazheng Xing   Group: iGEM15_Shenzhen_SFLS   (2015-08-26)
Revision as of 13:16, 17 September 2015 by VictorX (Talk | contribs)

The SCFV of anti-p185her2/neu antibody chA21 linked with EGFP by a (Gly4Ser)3 linker


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 370
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

The scFv CDS was synthesized by LCR assmebly using 36 oilgos

see oligos and protocols

The eGFP CDS was obtained using PCR. Primers: ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F) GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)

The eGFP CDS and the scFv CDS were connected by SOE PCR Primers: step1: CGGAATTCatgGGTTCTACTCAAG (SCFV-F) gctgcctccgcctccagaaccgccaccgccAGATTGACAATCTTCagaaga (SCFV-R) ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F) GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R) step2: SCFV-F and eGFP-R

Standard Biobrick prefix and suffix were then added to the sequence by PCR, replacing the EcoRI site and XbaI site which were originally designed for yeast expression. Primers: GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F) TACTAGTAGCGGCCGCTGCAGCTTGTACAGCTCGTCCAT(R)

Design Notes

References