Difference between revisions of "Part:BBa K1607011:Design"

(→‎Source)
(→‎Source)
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The eGFP CDS was obtained using PCR.
 
The eGFP CDS was obtained using PCR.
 
Primers:
 
Primers:
 +
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
 
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
  
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The eGFP CDS and the scFv CDS were connected by SOE PCR
 
The eGFP CDS and the scFv CDS were connected by SOE PCR
 +
 
Primers:
 
Primers:
 +
 
step1:
 
step1:
 +
 
CGGAATTCatgGGTTCTACTCAAG (SCFV-F)
 
CGGAATTCatgGGTTCTACTCAAG (SCFV-F)
  
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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.
 
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:
 
Primers:
 +
 
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)
 
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)
  

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