Difference between revisions of "Part:BBa K142000:Design"
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R197F forward | R197F forward | ||
| + | |||
CGGCGCGTCTGTTTCTGGCTGGCTG | CGGCGCGTCTGTTTCTGGCTGGCTG | ||
| + | |||
R197A forward | R197A forward | ||
| + | |||
CGGCGCGTCTGGCGCTGGCTGGCTG | CGGCGCGTCTGGCGCTGGCTGGCTG | ||
| + | |||
R197F reverse | R197F reverse | ||
| + | |||
CAGCCAGCCAGAAACAGACGCGCCG | CAGCCAGCCAGAAACAGACGCGCCG | ||
| + | |||
R197A reverse | R197A reverse | ||
| + | |||
CAGCCAGCCAGCGCCAGACGCGCCG | CAGCCAGCCAGCGCCAGACGCGCCG | ||
| + | |||
T276F forward | T276F forward | ||
| + | |||
GGATACGACGATTTTGAAGACAGCTC | GGATACGACGATTTTGAAGACAGCTC | ||
| + | |||
T276A forward | T276A forward | ||
| + | |||
GGATACGACGATGCGGAAGACAGCTC | GGATACGACGATGCGGAAGACAGCTC | ||
| + | |||
T276F reverse | T276F reverse | ||
| + | |||
GAGCTGTCTTCAAAATCGTCGTATCC | GAGCTGTCTTCAAAATCGTCGTATCC | ||
| + | |||
T276A reverse | T276A reverse | ||
| + | |||
GAGCTGTCTTCCGCATCGTCGTATCC | GAGCTGTCTTCCGCATCGTCGTATCC | ||
All lacI IS holding BioBricks were verified by sequencing. | All lacI IS holding BioBricks were verified by sequencing. | ||
| − | |||
===References=== | ===References=== | ||
Latest revision as of 14:46, 17 October 2008
lacI IS mutant (IPTG unresponsive) R197A
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
During site-directed mutagenesis, the codons to be mutated were replaced with the most highly utilized codons in E. coli to prevent complications from the use of rare codons. The lacI IS sequences were analyzed for BioBrick restriction sites within the coding sequence to ensure their compatibility.
Source
Source: The lacI IS mutants presented derive from the BioBrick holding lacI by itself (C0012). Site-directed mutagenesis was performed by PCR, subsequent DpnI digest and transformation. The following primers were used:
R197F forward
CGGCGCGTCTGTTTCTGGCTGGCTG
R197A forward
CGGCGCGTCTGGCGCTGGCTGGCTG
R197F reverse
CAGCCAGCCAGAAACAGACGCGCCG
R197A reverse
CAGCCAGCCAGCGCCAGACGCGCCG
T276F forward
GGATACGACGATTTTGAAGACAGCTC
T276A forward
GGATACGACGATGCGGAAGACAGCTC
T276F reverse
GAGCTGTCTTCAAAATCGTCGTATCC
T276A reverse
GAGCTGTCTTCCGCATCGTCGTATCC
All lacI IS holding BioBricks were verified by sequencing.