pTub-alpha1-EB3-NeonGreen-T2A-iLifeact-mCherry
(Plasmid #175261)

• Purpose: labelling of microtubule plus end and F-actin structures
• Depositing Labs: Frank Bradke, Sebastián Dupraz
• Publication: Alfadil et al J Vis Exp. 2021 Oct 14;(176). doi: 10.3791/63068.

Backbone

• Vector backbone: pTub-alpha1-EGFP
• Backbone size w/o insert (bp): 4492
• Vector type: Mammalian Expression

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: EB3-NeonGreen-T2A-iLifeact-mCherry
• Species: H. sapiens (human)
• Insert Size (bp): 2409
• Mutation: None (wt)
• Entrez Gene: MAPRE3 (a.k.a. EB3, EBF3, EBF3-S, RP3)
• Promoter: pTub-alpha1
• Tags / Fusion Proteins:
  • mNeonGreen (on EB3) (C terminal on insert)
  • mCherry (on iLifeact) (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: pTub-alpha1 ( 5'-GTAAGAAGCAACACCTCCTC-3')
• 3′ sequencing primer: mCherry reverse ( 5'-TTGGTCACCTTCAGCTTGG-3')

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • mNeonGreen Label License
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Improved (i)LifeAct-mCherry is prepared by mutating the start codon of the fluorophore ATGxCTG leaving a unique Kosak sequence-enhanced ATG start codon preceeding the LifeAct. We have experimentally detected in previous LifeAct versions that polymerase tend to skip LifeAct translation and start translation at the fluorophore instead, increasing fluorophore-derived background.