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

Latest revision as of 01:46, 2 November 2017

Standardized pBAD_Cas9 device

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1207
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.

@@ Line 21: / Line 21: @@
 ===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>