Difference between revisions of "Part:BBa K1983008:Design"
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===Csm4 mutation list=== | ===Csm4 mutation list=== | ||
| − | LA72F; LA236F; LA99F; YA143F; LA115F; LA219F; LA225F; LA191F; LA46F; YA258F; IA76F; YA114F; YA175F; LA235F; LA293F; LA25F; LA204F; YA234F; LA178F; LA106F; LA142F; LA20F; LA38F; IA7F; YA78F; IA193F; YA3F | + | LA72F; LA236F; LA99F; YA143F; LA115F; LA219F; LA225F; LA191F; LA46F; YA258F; IA76F; YA114F; YA175F; LA235F; LA293F; LA25F; LA204F; YA234F; LA178F; LA106F; LA142F; LA20F; LA38F; IA7F; YA78F; IA193F; YA3F |
===Primers=== | ===Primers=== | ||
Revision as of 13:20, 21 October 2016
Streptococcus thermofilus Csm4 phenylalanine mutant M27 with C-terminal 6XHis-Tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 796
Illegal XhoI site found at 907 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Designed including Esp3I restriction site before the start codon (ATG) for efficient cloning into expression vectors, XhoI site for cutting between the protein and 6XHis-Tag.
Note: Due to technical issues, the part's suffix in the backbone is changed by one FIRST letter (T to A). However, it does not remove or add any other restriction sites and does not change the function of the suffix. The purpose of this note is to alert false negative results during sequencing if the part is used in the future.
Original suffix: TACTAGTAGCGGCCGCTGCAG Part suffix: AACTAGTAGCGGCCGCTGCAG
Csm4 mutation list
LA72F; LA236F; LA99F; YA143F; LA115F; LA219F; LA225F; LA191F; LA46F; YA258F; IA76F; YA114F; YA175F; LA235F; LA293F; LA25F; LA204F; YA234F; LA178F; LA106F; LA142F; LA20F; LA38F; IA7F; YA78F; IA193F; YA3F
Primers
Primers used for amplification of the fragment:
Uni-FW: GTAGAATTCGCGGCCGCTTCTAG
Uni-RV: GTAGACTGCAGCGGCCGCTACTAG
Primers used for colony PCR screening:
For pETDuet-1:
Up-A1: GGATCTCGACGCTCTCCCT
Down3: ACCCCTCAAGACCCGTTTAG
Source
BBa_K1983008