sgFFL_d3
(Plasmid #59891)

• Purpose: synthetic autoregulatory gene circuit made by inserting an intron containing the mouse mir-124–3 gene into mCherry. Contains the miR-124-regulated 3′UTR of the Vamp3 gene with one seed site mutated.
• Depositing Lab: Georg Seelig
• Publication: Strovas et al ACS Synth Biol. 2014 May 16;3(5):324-31. doi: 10.1021/sb4001867. Epub 2014 Jan 30.

Backbone

• Vector backbone: pcDNA5 FRT/TO
• Vector type: Mammalian Expression
• Selectable markers: Hygromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: mCherry with intron containing the mouse mir-124–3
• Species: Synthetic
• Mutation: VAMP 3 UTR--3rd seed site removed
• Promoter: doxycycline (Dox)-inducible promoter (CMV/TO)
• Tags / Fusion Proteins:
  • PEST (C terminal on insert)
  • VAMP 3 UTR (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: mCherry-F
• 3′ sequencing primer: Bgh-rev

Resource Information

• Supplemental Documents:
  • sgFFL_d3 genbank file

Terms and Licenses

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

Depositor Comments

To create a repressive regulatory link between the miRNA and the mCherry transcript, we added a truncated version of the mir-124-regulated 3′UTR of the Vamp3 gene to the mRNA. This 3′UTR contains one 6-mer and three 7-mer target sites complementary to the mir-124 seed region (nucleotides 2−8 from the 5′ end of the miRNA). To better observe the dynamics of gene expression, we destabilized the mCherry protein using a standard PEST degradation tag.