Part:BBa_K1607011:Design

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)