Part:BBa_K4083000:Design

pRGPDuo2 plasmid for P. putida

Assembly Compatibility:

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

  Illegal NheI site found at 3853
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3880
  Illegal BamHI site found at 31
  Illegal XhoI site found at 2175
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6480
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Figure 1. Illustration of RPGDuo2 plasmid.

As illustrated in Figure 1, this plasmid contains two multiple cloning sites (MCS 1 and MCS2) with two transcriptional terminators. The MCS1 and MCS2 are regulated by IPTG-inducible Ptac and aTc-inducible PtetR/tetA promoters respectively. Moreover, there are lacI and tetR dependent repression systems that are regulated by Ptac and PtetR/tetA respectively. They were integrated for metabolic engineering purposes. There is a kanamycin resistance gene (KanR). There are two origins of replication to allow replication in E. coli (colE1) and P. putida (pRO1600).

Source

Modified from pRG_Duet for Corynebacterium glutamicum Gauttam et al.

References

[1] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514