Difference between revisions of "Part:BBa K2457005:Design"

 
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===Design Notes===
 
===Design Notes===
−
descrever
+
<b>Cas9</b>
−
 
+
The sequence was ordered as two gBlocks from Integrated DNA Technologies and it was assemble by PvuII restriction enzyme site.
−
 
+
<p>• NheI (GCTAGC), which in the alanine GCT codon (18% de codon usage) was replaced by GCG (32%)</p>
 +
<p>• EcoRI (C-GAATTC), which in arginine CGA codon (30%) was replaced by CGT (24.7%) </p>
 +
<p>• First HindIII (AAGCTT) changed to AAACTT</p>
 +
<p>• Second HindIII (AAGCTT) changed to AGGCTT</p>
 +
<p>• NdeI: CATATG changed to CACATG; CATATG changed to CATACG</p>
 +
<p>• NcoI: CCATGG changed to CCGTGG</p>
 +
<p>• BamHI: GGATCC changed to GGACCC</p>
  
 
===Source===
 
===Source===
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<b>Cas9</b> M1GAS Streptococcus pyogenes
−
descrever
+
  
 
===References===
 
===References===
 +
<p>Nishimasu, Hiroshi; Hsu, Patrick D.; Shehata, Soraya; Nureki, Osamu. et al.  2014. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5): 935–949.</p>
 +
<p>Church, George M.; Mali, Prashant; Esvelt, Kevin M. 2013. Cas9 as a versatile tool for engineering biology. Nature Methods, 10: 957-963.</p>

Latest revision as of 01:46, 2 November 2017


Standardized pBAD_Cas9 device


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1042
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1024


Design Notes

Cas9 The sequence was ordered as two gBlocks from Integrated DNA Technologies and it was assemble by PvuII restriction enzyme site.

• NheI (GCTAGC), which in the alanine GCT codon (18% de codon usage) was replaced by GCG (32%)

• EcoRI (C-GAATTC), which in arginine CGA codon (30%) was replaced by CGT (24.7%)

• First HindIII (AAGCTT) changed to AAACTT

• Second HindIII (AAGCTT) changed to AGGCTT

• NdeI: CATATG changed to CACATG; CATATG changed to CATACG

• NcoI: CCATGG changed to CCGTGG

• BamHI: GGATCC changed to GGACCC

Source

Cas9 M1GAS Streptococcus pyogenes

References

Nishimasu, Hiroshi; Hsu, Patrick D.; Shehata, Soraya; Nureki, Osamu. et al. 2014. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5): 935–949.

Church, George M.; Mali, Prashant; Esvelt, Kevin M. 2013. Cas9 as a versatile tool for engineering biology. Nature Methods, 10: 957-963.