Device

Part:BBa_K863204:Design

Designed by: Julia Schirmacher   Group: iGEM12_Bielefeld-Germany   (2012-09-23)

shuttle vector pECPP11JS for recombination of genes of interest in yeast


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 4086
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3381
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
    Illegal BglII site found at 1532
    Illegal BamHI site found at 1889
    Illegal XhoI site found at 1196
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 4086
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 4086
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4242
    Illegal BsaI.rc site found at 2041


Design Notes

This part was build by a Gibson assembly. 5' overlap parts were created by PCR with the following primers.

The 5' UTR of alcohol oxidase 1 gene (AOX1) of Pichia pastoris was created with the primers:

FW: 5'-CGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGAGATCTAACATCCAAAGACG-3'

RV: 5'-GGTGGCGGCGGGCGTTTCGAATAATTAGTT-3'


The 3' UTR of alcohol oxidase 1 gene (AOX1) of Pichia pastoris was created with the primers:

FW: 5'-TTATTTAGAGATTTTAACTTACATTTAGATTCGATAGATCCGAGTATCTATGATTGGAAG-3'

RV: 5'-AAAACAAGATAGTGCCCCTC-3'


The his4 gene from Pichia pastoris was created with the primers:

FW: 5'-GTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTAAGATCTCCTGATGACTGACTC-3'

RV: 5'-CTCGGATCTATCGAATCTAAATGTAAG-3'


The mating factor alpha 1 with Kozak sequence was created with the primers:

FW: 5'-AGAAGATCAAAAAACAACTAATTATTCGAAACGCCCGCCGCCACCATGAGATTTCCTTCAATTTTTAC-3'

RV: 5'-AGCTTCAGCCTCTCTTTTCT-3'


The AOX1 Terminator from Pichia pastoris was created with the primers:

FW: 5'-GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACACGCAGGTGGTATGTATCACCTGCGTGTCTTGCTAGATTCTAATCAAG-3'

RV: 5'-TAAGCTTGCACAAACGAACT-3'


All components were created as BioBricks for the community:

  • 5' UTR of alcohol oxidase 1 gene (AOX1) of Pichia pastoris K863200
  • 3' UTR of alcohol oxidase 1 gene (AOX1) of Pichia pastoris K863201
  • his4 gene from Pichia pastoris K863202
  • mating factor alpha 1 with Kozak sequence K863206
  • AOX1 Terminator from Pichia pastoris K863203


By site directed mutagenesis the XbaI restriction site have to be deleted.

Source

The wild type P. pastoris X-33 and pPICzA from [http://products.invitrogen.com/ivgn/product/V19020 Invitrogen].

The gene sequence of TVEL5 was originally isolated from Trametes versicolor. We got the cDNA plasmid sequence from a working group from the Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald (Germany).

References