Part:BBa_K1045014:Design

Promoter reverse - Promoter - DarR operator - GFP generator BBa_E0240

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1
  Illegal NheI site found at 24
  Illegal NheI site found at 104
  Illegal NheI site found at 127
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 55
  Illegal AgeI site found at 963
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 825
  

Design Notes

This part was constructed using hybridization oligos for BBa_K1045011. The hybridization product corresponded to a DNA fragment harboring the sequence of BBa_K1045011 cut with EcoRI and SpeI at the prefix and suffix sites. This fragment was ligated into BBa_K1045013 in a prefixing composition.

This biobrick harbors a mutation in the suffix sequence. The mutated suffix sequence is: TACTAGTAACGGCCGCTGCAG. This eliminates the NotI restriction site. The plasmid might still be cut with SpeI and PstI.

Source

The hybridization oligos mentioned under "Design Notes" were purchased from Sigma-Aldrich. The sequence of the promoter originated from the Parts Registry page of BBa_J23117. The 54 bp upstream of the promoter represent a random sequence which would translate into "cyclicdiampacterim".

BBa_K1045013 originated from parts of the distribution kit 2013 and from hybridization oligos. The sequence information for hybridization oligos was obtained from the parts registry and from Zhang et al., 2013. The hybridization oligos were purchased from Sigma-Aldrich.

References

Lei Zhang, Weihui Li, and Zheng-Guo He (2013) “DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis”, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 5, pp. 3085–3096