Difference between revisions of "Part:BBa K863204:Design"
JSchirmacher (Talk | contribs) (→Source) |
JSchirmacher (Talk | contribs) (→Design Notes) |
||
Line 50: | Line 50: | ||
* mating factor alpha 1 with Kozak sequence [https://parts.igem.org/Part:BBa_K863206 K863206] | * mating factor alpha 1 with Kozak sequence [https://parts.igem.org/Part:BBa_K863206 K863206] | ||
* AOX1 Terminator from Pichia pastoris [https://parts.igem.org/Part:BBa_K863203 K863203] | * AOX1 Terminator from Pichia pastoris [https://parts.igem.org/Part:BBa_K863203 K863203] | ||
+ | |||
+ | |||
+ | By site directed mutagenesis the ''Xba''I restriction site have to be deleted. | ||
===Source=== | ===Source=== |
Revision as of 03:45, 27 September 2012
shuttle vector pECPP11JS for recombination of genes of interest in yeast
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 4086
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3381
- 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 4086
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 4086
- 1000INCOMPATIBLE 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].