Part:BBa_K5487114:Design

MCS Box-lac operator-lac promoter-CAP binding site

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 72
  Illegal XbaI site found at 45
  Illegal PstI site found at 33
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 72
  Illegal PstI site found at 33
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 72
  Illegal BamHI site found at 51
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 72
  Illegal XbaI site found at 45
  Illegal PstI site found at 33
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 72
  Illegal XbaI site found at 45
  Illegal PstI site found at 33
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Obtained after synthesizing plasmid PHCY163 at Bio, transformed and amplified within E. coli DH5α

Source

M13 fwd and M13 rev Source: These are primer binding sites derived from the M13 bacteriophage, a filamentous coliphage that infects bacteria like Escherichia coli. The M13 phage is often used in molecular biology for single-stranded DNA production, and its sequences are utilized in universal primers for DNA sequencing and PCR applications. Multiple Cloning Site (MCS) Source: The MCS does not originate from a specific natural genomic sequence. Instead, it is a synthetic construct that has been engineered to contain a series of unique restriction sites. These sites are selected from various sources and assembled in a sequence that allows for the convenient cloning of DNA fragments. lac Operator and lac Promoter Source: Both the lac operator and the lac promoter are derived from the lac operon of Escherichia coli. The lac operon is a well-studied part of bacterial DNA that regulates the metabolism of lactose, a sugar found in milk. The operon includes genes that code for proteins responsible for the transport and breakdown of lactose in bacterial cells. CAP Binding Site Source: The catabolite activator protein (CAP) binding site is also naturally occurring in Escherichia coli. It is a regulatory sequence found near promoters of certain operons like the lac operon. CAP, when bound to cyclic AMP (cAMP), enhances the expression of genes involved in catabolizing alternative sugar sources when glucose is scarce, including those in the lac operon.

References

Huang, C., Guo, L., Wang, J. et al. Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering. Appl Microbiol Biotechnol 104, 7943–7956 (2020). https://doi.org/10.1007/s00253-020-10819-1