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

 
(→‎Design Notes)
Line 10: Line 10:
  
 
   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 pPICZα-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.
 
   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 pPICZα-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.
−
 
−
 
  
 
===Source===
 
===Source===

Revision as of 04:54, 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]


Design Notes

The restriction sites: 

  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 pPICZα-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.

Source

The obtainment of DNA sequence: 

  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.


References