hGFAP-GFP
(Plasmid #40592)

• Depositing Lab: Albee Messing
• Publication: Zhuo et al Dev Biol. 1997 Jul 1;187(1):36-42.

Backbone

• Vector backbone: pgfa2-lac2
• Backbone manufacturer: Michael Brenner (PMID: 7952266)
• Backbone size w/o insert (bp): 5500
• Total vector size (bp): 6250
• Modifications to backbone: LacZ coding region of pgfa2-lac2 replaced by a mutated, humanized hGFP(S65T) cDNA.
• Vector type: Mammalian Expression, Bacterial Expression, Mouse Targeting

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: GFP-S65T
• Alt name: hGFP-S65T
• Alt name: GFP
• Alt name: Tg94.7
• Species: H. sapiens (human), M. musculus (mouse), Synthetic; Aequorea victoria (humanized)
• Insert Size (bp): 750
• Mutation: Construct contains a mutated, humanized hGFP(S65T) cDNA followed by a heterologous mouse intron and polyadenylation signal, all under the control of the gfa2 promoter
• Promoter: hGFAP promoter
• Tag / Fusion Protein:
  • MP-1 fragment (mouse protamine 1 intron and polyadenylation signal) (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (not destroyed)
• 3′ cloning site: XbaI (unknown if destroyed)
• 5′ sequencing primer: GFAPpro-F (5'-ACTCCTTCATAAAGCCCTCG-3')

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Dr. Brenner (GFAP/MP-1 backbone)
• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This plasmid encodes a transgene to express hGFP-S65T under the control of the hGFAP promoter in transgenic mice. The plasmid Gfa2-lac2, containing the human GFAP promoter and mouse protamine 1 intron and polyadenylation signal on the 3' end, was kindly provided by Dr. M. Brenner of the NIH. The plasmid phGFP-S65T containing the mutated, humanized GFP cDNA was purchased from the Clonetech Laboratories. The transgene was constructed by excising the lacZ coding region from the pGfa2-lac2 plasmid by BamHI digestion, and replacing it by blunt end ligation with a 0.75-kb HindIII-XbaI fragment containing the entire GFP coding region from phGFP-S65T.

hGFP-S65T is a variant of the Aequorea victoria green fluorescent protein that has been optimized for brighter fluorescence and higher expression in human (and mammalian) cells. hGFP-S65T contains more than 190 silent base changes to optimize the coding sequence based on human codon-usage preferences. The S65T mutation results in a single, red-shifted excitation peak at 490 nm and brighter fluorescence.