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tion tags like His6-tag on the same side as the inhibitor. This
ensures that if truncated fragments are present in the cell lysate,
it is the inhibitor and not free enzyme which is pulled down.
Free enzyme can lead to a high background.
3. The DNA sequence coding for Tem1-BLIP is given below. For
expression, it may be inserted into pET28a NdeI XhoI sites:
5′-aaaaaaCATATGCACCCGGAAACCCTGGTTAAAGTCA
AAGATGCCGAAGACCAGCTGGGTGCACGCGTTGGC
TATATTGAACTGGATCTGAACTCAGGCAAAATCCTG
GAATCGTTTCGTTCTGAAGAACGCTTCCCGATG
AT G T C A A C C T T TA A A G T T C T G C T G T G C G G T G
CTATCCTGTCGCGTATTGATGCGGGTCAGGAA
CAACTGGGCCGTCGCATCCATTATTCACAGAAT
GATCTGGTCGAATACTCGCCGGTGACCGAAAAA
CACCTGACCGACGGCATGACGGTGCGCGAACTGT
GTAGCGCCGCAATTACCATGTCTGATAACACGGCT
G C G A A T C T G C T G C T G A C C A C G A T C G G T G G T
CCGAAAGGCCTGACCGCCTTCCTGCATAACATGGG
T G AT C A C G T TA C G C G T C T G G A C C G C T G G G A A
C C G G A A C T G A A C G A A G C C A T T C C G A A T G A
TGAACGTGACACCACGACCCCGGTGGCAATGG
CAACCACGCTGCGCAAACTGCTGACCGGCGAAC
T G C T G A C G C C G G C G A G T C G T C A G C A A C T G A
TGGATTGGATGGAAGCCGACAAAGTGGCAGGT
C C G C T G C T G C G TA G C G T G C T G C C G G C A G G T T
G G T T TAT C G C A G ATA A A A G T G G T G C A G G C G A A
C G T G G T T C C C G C G G C A T T G T G G C T G C A C
T G G G T C C G G A T G G C A A A C C G A G T C G C A T T
G T G G T TAT C TATA C C A C G G G C T C C C A G G C G A
C C A T G G A T G A A C T G A A T C G C C A A A T T G
C T G A A A T C G G T G C G A G C C T G A T C A A A C A T
T G G G G C G G T A G T G G T G G C G A A A A A A T T C G
T C T G C G T G G A T C C G C T G G T G T T A T G A C C G G
C G C G A A A T T T A C G C A G A T T C A A T T C G G T A T
GACCCGTCAGCAAGTCCTGGATATCGCCGGCG
CAGAAAACTGCGAAACCGGCGGTAGCTTTGGT
GACTCTATTCATTGTCGTGGTCACGCAGCAGGT
GCATATTACGCGTATGCAACCTTTGGCTTCACG
AGCGCAGCTGCGGATGCCAAAGTGGACTCAAAAT
CGCAGGAAAAACTGCTGGCTCCGTCTGCACCGACC
CTGACGCTGGCAAAATTCAATCAGGTCACCGTGGGT
ATGACGCGTGCTCAGGTGCTGGCGACCGTTGG
TCAAGGCAGTTGCACCACGTGGTCCGAATATTAC
CCGGCTTACCCGAGCACCGCAGGTGTGACGCTGA
GTCTGTCTTGTTTTGATGTTGACGGTTATAGCTCT
ACCGGTTTCTACCGTGGCTCCGCGCATCTGTGGTTT
ACGGATGGTGTCCTGCAGGGCAAACGCCAATGGGA
CCTGGTGGGCGGTGGGAGCTCCTATCCGTACGAT
GTTCCGGACTACGCCTAACTCGAGaaaaaa- 3′.
Sensing by Allosteric Derepression
tion tags like His6-tag on the same side as the inhibitor. This
ensures that if truncated fragments are present in the cell lysate,
it is the inhibitor and not free enzyme which is pulled down.
Free enzyme can lead to a high background.
3. The DNA sequence coding for Tem1-BLIP is given below. For
expression, it may be inserted into pET28a NdeI XhoI sites:
5′-aaaaaaCATATGCACCCGGAAACCCTGGTTAAAGTCA
AAGATGCCGAAGACCAGCTGGGTGCACGCGTTGGC
TATATTGAACTGGATCTGAACTCAGGCAAAATCCTG
GAATCGTTTCGTTCTGAAGAACGCTTCCCGATG
AT G T C A A C C T T TA A A G T T C T G C T G T G C G G T G
CTATCCTGTCGCGTATTGATGCGGGTCAGGAA
CAACTGGGCCGTCGCATCCATTATTCACAGAAT
GATCTGGTCGAATACTCGCCGGTGACCGAAAAA
CACCTGACCGACGGCATGACGGTGCGCGAACTGT
GTAGCGCCGCAATTACCATGTCTGATAACACGGCT
G C G A A T C T G C T G C T G A C C A C G A T C G G T G G T
CCGAAAGGCCTGACCGCCTTCCTGCATAACATGGG
T G AT C A C G T TA C G C G T C T G G A C C G C T G G G A A
C C G G A A C T G A A C G A A G C C A T T C C G A A T G A
TGAACGTGACACCACGACCCCGGTGGCAATGG
CAACCACGCTGCGCAAACTGCTGACCGGCGAAC
T G C T G A C G C C G G C G A G T C G T C A G C A A C T G A
TGGATTGGATGGAAGCCGACAAAGTGGCAGGT
C C G C T G C T G C G TA G C G T G C T G C C G G C A G G T T
G G T T TAT C G C A G ATA A A A G T G G T G C A G G C G A A
C G T G G T T C C C G C G G C A T T G T G G C T G C A C
T G G G T C C G G A T G G C A A A C C G A G T C G C A T T
G T G G T TAT C TATA C C A C G G G C T C C C A G G C G A
C C A T G G A T G A A C T G A A T C G C C A A A T T G
C T G A A A T C G G T G C G A G C C T G A T C A A A C A T
T G G G G C G G T A G T G G T G G C G A A A A A A T T C G
T C T G C G T G G A T C C G C T G G T G T T A T G A C C G G
C G C G A A A T T T A C G C A G A T T C A A T T C G G T A T
GACCCGTCAGCAAGTCCTGGATATCGCCGGCG
CAGAAAACTGCGAAACCGGCGGTAGCTTTGGT
GACTCTATTCATTGTCGTGGTCACGCAGCAGGT
GCATATTACGCGTATGCAACCTTTGGCTTCACG
AGCGCAGCTGCGGATGCCAAAGTGGACTCAAAAT
CGCAGGAAAAACTGCTGGCTCCGTCTGCACCGACC
CTGACGCTGGCAAAATTCAATCAGGTCACCGTGGGT
ATGACGCGTGCTCAGGTGCTGGCGACCGTTGG
TCAAGGCAGTTGCACCACGTGGTCCGAATATTAC
CCGGCTTACCCGAGCACCGCAGGTGTGACGCTGA
GTCTGTCTTGTTTTGATGTTGACGGTTATAGCTCT
ACCGGTTTCTACCGTGGCTCCGCGCATCTGTGGTTT
ACGGATGGTGTCCTGCAGGGCAAACGCCAATGGGA
CCTGGTGGGCGGTGGGAGCTCCTATCCGTACGAT
GTTCCGGACTACGCCTAACTCGAGaaaaaa- 3′.
Sensing by Allosteric Derepression