pCMVd1-NLS-GAL4-iLight-VP16-T2A-mTagBFP2
(Plasmid #170273)

• Purpose: NLS-Gal4(1-65)-iLight(human codon optimized)-VP16-T2A-mTagBFP2 construct expression in mammalian cells.
• Depositing Lab: Vladislav Verkhusha
• Publication: Kaberniuk et al Nat Commun. 2021 Jun 23;12(1):3859. doi: 10.1038/s41467-021-24212-7.

Backbone

• Vector backbone: pQP-Select
• Backbone manufacturer: Originally derived from pEGFP-N1 backbone after several modifications in our lab.
• Backbone size w/o insert (bp): 3427
• 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: NLS-Gal4(1-65)-iLight(human codon optimized)-VP16-T2A-mTagBFP2
• Species: Bradyrhizobium sp. (strain ORS 278)
• Insert Size (bp): 3154
• Mutation: See Comments
• Promoter: CMVd1

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: CMV Forward
• 3′ sequencing primer: EBV Reverse

Resource Information

• Supplemental Documents:
  • pCMVd1-NLS-GAL4-iLight-VP16-T2A-mTagBFP2.gb

Terms and Licenses

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

Depositor Comments

To design iLight, a full-length bacterial phytochrome IsPadC from Idiomarina sp. A28L was used as an original template. First, diguanylyl cyclase effector in IsPadC was removed, then following mutations were introduced: I68F, H80Q, A86T, R90S S242C, E274K, R295H I360V, and L464V. After that, iLight was human codon optimized for mammalian expression.