Difference between revisions of "Part:BBa K1607011:Design"
(→‎Source)
Line 13: Line 13:
 
Primers:
 
Primers:
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
 +
 
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
 
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
  
Line 19: Line 20:
 
step1:
 
step1:
 
CGGAATTCatgGGTTCTACTCAAG (SCFV-F)
 
CGGAATTCatgGGTTCTACTCAAG (SCFV-F)
 +
 
gctgcctccgcctccagaaccgccaccgccAGATTGACAATCTTCagaaga (SCFV-R)
 
gctgcctccgcctccagaaccgccaccgccAGATTGACAATCTTCagaaga (SCFV-R)
 +
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
 +
 
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
 
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
 +
 
step2:
 
step2:
 +
 
SCFV-F and eGFP-R
 
SCFV-F and eGFP-R
 
   
 
   
Line 28: Line 34:
 
Primers:
 
Primers:
 
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)
 
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)
 +
 
TACTAGTAGCGGCCGCTGCAGCTTGTACAGCTCGTCCAT(R)
 
TACTAGTAGCGGCCGCTGCAGCTTGTACAGCTCGTCCAT(R)
 +
 
===Design Notes===
 
===Design Notes===
  
 
===References===
 
===References===

Revision as of 13:19, 17 September 2015

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