PSD-95_Y12E-pTagRFP
(Plasmid #52672)

• Purpose: expression of TagRFP-tagged PSD-95_Y12E
• Depositing Lab: Johannes Hell
• Publication: Hell lab plasmids (unpublished)

Backbone

• Vector backbone: pTagRFP
• Backbone manufacturer: Evrogen
• Backbone size w/o insert (bp): 4733
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: PSD-95
• Alt name: Postsynaptic density protein 95
• Alt name: Disks large homolog 4 (DLG4)
• Alt name: Synapse-associated protein 90 (SAP-90)
• Species: R. norvegicus (rat)
• Mutation: Y12E
• GenBank ID: P78352
• Entrez Gene: Dlg4 (a.k.a. Dlgh4, PSD95, Sap90)
• Promoter: CMV
• Tag / Fusion Protein:
  • tagRFP (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: mKate2-TurboRFP-R

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

HindII and EcoRI were used to cut PSD-95 from PSD-95-pGW1 (received from David Bredt) and ligate it into pTagRFP-N. PSD-95 tyrosine 12 was mutated to glutamate (Y12E) by extending a complementary set of primers (PSD95Y12E-forward: 5’-GAC AAC CAA GAA AGA GCG CTA CCA AGA TGA AGA CAC G-3’, PSD95Y12E-reverse: 5'-CGT GTC TTC ATC TTG GTA GCG CTC TTT CTT GGT TGT C-3') along the whole template plasmid using Q5 high-fidelity DNA polymerase (NEB). DpnI was subsequently used to digest non-mutated, methylated template DNA prior to transformation.