Part:BBa_K3757011:Design
3in1 Oak1 CtAEP1 sGFP (S65T)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2239
Illegal NheI site found at 4838
Illegal NheI site found at 6434
Illegal NheI site found at 6677 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4142
Illegal BamHI site found at 1577
Illegal BamHI site found at 1967
Illegal BamHI site found at 4398
Illegal BamHI site found at 4569 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 91
Illegal BsaI site found at 175
Illegal BsaI site found at 2326
Illegal BsaI site found at 4925
Illegal BsaI.rc site found at 6733
Design Notes
The part was assembled with Golden Gate assembly; an arbitrary peptide to cyclize can be inserted into the Oak1 precursor in a single Esp3I type IIS restriction enzyme mediated cloning step, and correct clones may be selected with blue-white-selection; the DNA sequence is codon-optimized for Nicotiana benthamiana.
Source
Oak1 (BBa_K3757001) from Oldenlandia affinis, sequence from GenBank: AF393825.1 MCoTI-II_loop5_His6 (BBa_K3757002) from Momordica cochinchinensis. Amino acid sequence from Cybase (http://www.cybase.org.au/index.php?page=card&table=protein&id=3) reverse transcibed into DNA sequence with codon usage optimized for Nicotiana bethaniana. c-myc tag originates from the c-myc gene, provided in Golden Gate vector. CtAEP1 from Clitoria ternatea, sequence found through Genbank: KF918345.1 HA-tag originates from the the human influenza virus hemagglutinin protein, provided in Golden Gate vector. GFP originates from Aequorea victoria, sGFP (S65T) mutant with improved fluorescent properties, obtained in lower-level Golden Gate vectors. 35S promoter and terminator originate from Cauliflower mosaic virus, obtained in lower-level Golden Gate vectors.