Part:BBa_K3878000

pET24d(+) + GFP

This part is a pET24d(+) carrying EGFP coding sequence followed by a T7 terminator. pET_24d(+)_GFP is kanamycin resistant. This part is the Host plasmid for the different sensors targeting the miRNA 141 (Part BBa_K3878001, BBa_K3878008-12). We took an NEB pET-24(d)+ with a kanamycin cassette. We digest it through two restriction enzymes EcoRI and Xho1 and insert EGFP in between [1,2].

This cloning vector is ready to receive toehold switches without the repressed gene which is here EGFP. The insert must be preceded by a T7 promoter.

Usage and Biology

This plasmid contains T7 promoter and Terminator, in order to be used in E.Coli containing the T7 RNA Polymerase. The reporter Gene is EGFP which is a derivative of the Green Fluorescent Protein from Aequorea victoria. It has been reported to be a rapidly-maturing weak dimer with moderate acid sensitivity. This part allows rapid digestion and insertion of different sequences. Restriction site before EGFP contains a T7 promoter, if you plan to insert a sequence before EGFP we strongly advise that you add your own T7 promoter sequence inside.

Figure 1 : Plasmid Map of the pET-24d(+)+EGFP. Created with SnapGene.

On Figure 1 you can see the various restriction you can use in order to put your sequence before EGFP. Pay attention to the restriction site to avoid homology sequence leading to plasmid closure before ligation.

References

[1] Li L, Pang D, Wang T, Li Z, Chen L, Zhang M, Song N, Nie D, Chen Z, Lai L, Ouyang H. Production of a reporter transgenic pig for monitoring Cre recombinase activity. Biochem Biophys Res Commun. 2009 May 1;382(2):232-5. doi: 10.1016/j.bbrc.2009.02.146. Epub 2009 Mar 5. PMID: 19268654.

[2] Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33-8. doi: 10.1016/0378-1119(95)00685-0. PMID: 8707053.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 5030
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 5074
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4964
  Illegal BamHI site found at 5105
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 5030
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 5030
  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 3184
  Illegal NgoMIV site found at 3344
  Illegal NgoMIV site found at 4932
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2263