pCMV(MinDis).iGluSnFR
(Plasmid #41732)

• Purpose: Single-wavelength extracellular glutamate sensor constructed from E. coli Gltl and cpGFP. Membrane-displayed. ** A newer version of this sensor is available. Please see iGluSnFR3 plasmids at https://www.addgene.org/browse/article/28220233/ **
• Depositing Lab: Loren Looger
• Publication: Marvin et al Nat Methods. 2013 Feb;10(2):162-70. doi: 10.1038/nmeth.2333. Epub 2013 Jan 13.

Backbone

• Vector backbone: pCMV(MinDis) [variant of pDisplay]
• Backbone manufacturer: modified from Invitrogen
• Backbone size w/o insert (bp): 5300
• Total vector size (bp): 6829
• Modifications to backbone: From pDisplay: Hemagglutinin (HA) tag removed
• Vector type: Mammalian Expression ; Glutamate Biosensor
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: GltI based Glutamate Biosensor
• Alt name: iGluSnFR
• Alt name: gltI/ybeJ
• Alt name: cpGFP146
• Species: Synthetic; E. coli
• Insert Size (bp): 1570
• Mutation: cpGFP146 variant from EcMBP165-cpGFP.PPYF.pRSET inserted between AA253-254 of E. coli gltI/ybeJ
• Promoter: CMV
• Tags / Fusion Proteins:
  • mouse Ig κ-chain (N terminal on backbone)
  • Myc (C terminal on backbone)
  • PDGFR transmembrane helix (aa 513-561) (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BglII (not destroyed)
• 3′ cloning site: PstI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: BGH-rev

Resource Information

• Supplemental Documents:
  • YbeJ253.L1LVL2NP.pMinDis.pdf
  • YbeJ253.L1LVL2NP.pMinDis.sbd
  • YbeJ253.L1LVL2NP.pMinDis_1.gb
• A portion of this plasmid was derived from a plasmid made by: E. coli glutamate-binding protein (gltI) from FLIP-E (Okumoto, et al. 2005, PNAS, PMID: 15939876) and cpGFP146 variant from EcMBP165-cpGFP.PPYF.pRSET (Addgene plasmid #33372)
• Articles Citing this Plasmid:
  • 16 References

Terms and Licenses

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

Depositor Comments

** A newer version of this sensor is available. Please see iGluSnFR3 plasmids at https://www.addgene.org/browse/article/28220233/ **

Biosensor iGluSnFRa is a single-wavelength glutamate sensor constructed from E. coli GltI and cpGFP. iGluSnFR is bright and photostable, with 4.5 (ΔF/F)max in vitro, under both 1- and 2-photon illumination.

pCMV(MinDis).iGluSnFR was constructed by subcloning the GltI253-cpGFP.L1LV/L2NP gene from pRSET.GltI253-cpGFP.L1LV/L2NP (Addgene plasmid #41733) into the pDisplay vector (Invitrogen) between the BglII and PstI sites. The hemagglutinin (HA) tag was removed by PCR, generating the backbone variant dubbed pCMV(MinDis).

This construct encodes an N-terminal mouse immunoglobin κ-chain leader sequence, which directs the protein to the secretory pathway; the GltI253.L1LV/L2NP protein; a myc epitope tag; and at the C-terminus, a platelet derived growth factor receptor transmembrane helix, which anchors the protein to the plasma membrane, displaying it on the extracellular side.

Detailed amino acid sequences are provided in the supplementary documents available above.

Alternate plasmid names: YbeJ253.L1LVL2NP.pMinDis; pMinDis.iGluSnFR