Part:BBa_K5375001

pA7-GFP

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BamHI site found at 4127
  Illegal XhoI site found at 3302
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 4091
  

Origin

Synthesized by company

Properties

Expression of Green Fluorescent Protein (GFP)

Usage and Biology

Green Fluorescent Protein (GFP) is a bioluminescent protein initially isolated from the jellyfish *Aequorea victoria*, characterized by its distinctive biological properties and extensive applications. GFP exhibits spontaneous green fluorescence without the necessity for any substrates or cofactors, rendering it an invaluable tool in biological research. In cell biology and molecular biology, GFP is frequently employed as a reporter gene through fusion with the coding sequence of target proteins, enabling real-time monitoring of their localization, dynamic alterations, and expression patterns within live cells. Furthermore, GFP can be utilized to label specific cellular organelles or structures, assisting researchers in observing cellular processes such as protein transport, signal transduction pathways, and cell division. Owing to its stability and ease of detection, GFP has become an essential component of contemporary life sciences research and has significantly advanced our understanding of cellular biological mechanisms.

Cultivation and Purification

We performed pA7-GFP plasmid linearization by enzyme digest and electrophoresis. This digest DNA fragment was utilized for ligation with the target gene.

Figure 1. The vector of pA7-GFP was enzyme digested.

Measurement and Characterization

To obtain the fusion protein and its expression, we constructed plasmids pA7-PFN3-GFP and pA7-HSP70-GFP. Validation was conducted by Sanger sequencing, as illustrated in the following figures:

Figure 2. Sanger sequencing map of pA7-GFP-PFN3 and pA7-GFP-HSP70.

Reference

Chalfie M., Tu Y., Euskirchen G., Ward W. W., & Prasher D. C. (1994). Green fluorescent protein as a marker for gene expression. *Science*, 263(5153), 802-805. https://doi.org/10.1126/science.8333281

Cava F., de Pedro M. A., Blas-Galindo E., Waldo G. S., Westblade L. F., & Berenguer J. (2008). Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology. *Environmental Microbiology*, 10(3), 605–613. https://doi.org/10.1111/j.1462-2920.2007.01482.x