Difference between revisions of "Part:BBa K1607010:Design"

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__NOTOC__
 
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<partinfo>BBa_K1607010 short</partinfo>
 
<partinfo>BBa_K1607010 short</partinfo>
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<partinfo>BBa_K1607010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1607010 SequenceAndFeatures</partinfo>
  
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===Source===
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This coding sequence was synthesized by LCR assmebly using 36 oilgos
  
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===Design Notes===
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[[see oligos and protocols]]
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The restriction sites:
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  The 601bp sequence synthesized by LCR assembly actually included an EcoRI site and an XbaI site at the ends of it. These two restriction sites were designed for pPICZ&#945;-a yeast expression vector, but the yeast expression plan was eventually given up due to its complexity. After the LCR assembly, standard Biobrick prefix and suffix were added to the sequence by PCR, replacing the originally designed EcoRI and XbaI. Finally, the standard part was cloned to PSB1C3 vector.
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===Design note===
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'''Isolation:'''
  
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===Source===
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After the LCR assembly, we isolated the CDS using these two primers(with BioBrick Prefix in the fwd primer and Suffix in the rev primer):
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fwd:GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt
  
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The obtainment of DNA sequence:  
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rev:TACTAGTAGCGGCCGCTGCAGAGATTGACAATCTTCAGAAGAT
  
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  To get this coding sequence, we found the AA sequence of this SCFV on NCBI first (PDB: 3H3B_B). The AA sequence was then reverse translated into DNA sequence. The DNA sequence was analyzed by gene2oligo and 36 oligos were given as a result. We synthesized the 36 oligos and obtained the 601bp fragment of SCFV coding sequence by LCR assembly using these oligos.
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'''The termination codon:'''
  
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Since it was originally designed for 3'his-tag vectors, this coding sequence does not have a termination codon.
  
 
===References===
 
===References===
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[1]Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.

Latest revision as of 06:02, 24 August 2015

The coding sequence of the SCFV of anti-p185her2/neu antibody chA21


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

This coding sequence was synthesized by LCR assmebly using 36 oilgos

see oligos and protocols

Design note

Isolation:

After the LCR assembly, we isolated the CDS using these two primers(with BioBrick Prefix in the fwd primer and Suffix in the rev primer):

fwd:GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt

rev:TACTAGTAGCGGCCGCTGCAGAGATTGACAATCTTCAGAAGAT

The termination codon:

Since it was originally designed for 3'his-tag vectors, this coding sequence does not have a termination codon.

References

[1]Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.