Composite

Part:BBa_K3757011:Design

Designed by: Erik Zimmer   Group: iGEM21_Tuebingen   (2021-09-29)


3in1 Oak1 CtAEP1 sGFP (S65T)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

References