pDR111_mannose_Klf4Gata-3
(Plasmid #188401)

• Purpose: Expresses mouse transcription factors Klf4 and Gata-3 in an operon from D-mannose promoter for Bacillus subtilis. The secretion peptide PhoD is fused to both factors.
• Depositing Lab: Christopher Contag
• Publication: Madsen et al Commun Biol. 2022 Aug 30;5(1):888. doi: 10.1038/s42003-022-03851-6.

Backbone

• Vector backbone: pDR111
• Backbone size w/o insert (bp): 8858
• Total vector size (bp): 11995
• Modifications to backbone: Addition of insert and fusion components to NheI site. Phyper-spank replaced by D-mannose promoter and lacI replaced by mannose regulator
• Vector type: Bacterial Expression
• Selectable markers: Spectinomycin for Bacillus subtilis

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Klf4
• Species: M. musculus (mouse)
• Insert Size (bp): 1452
• Mutation: See Depositor Comments Below
• Entrez Gene: Klf4 (a.k.a. EZF, Gklf, Zie)
• Promoter: D-mannose
• Tag / Fusion Protein:
  • PhoD secretion peptide (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: SbfI (not destroyed)
• 3′ cloning site: EagI (not destroyed)
• 5′ sequencing primer: caatcggctagcTTAGCCCATAG
• 3′ sequencing primer: tcgactCCTGCAGGaggaaac

Gene/Insert 2

• Gene/Insert name: Gata-3
• Species: M. musculus (mouse)
• Insert Size (bp): 1330
• Entrez Gene: Gata3 (a.k.a. Gata-3, jal)
• Promoter: D-mannose
• Tag / Fusion Protein:
  • PhoD secretion peptide

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: EagI (not destroyed)
• 3′ cloning site: NheI (not destroyed)
• 5′ sequencing primer: caatcggctagcTTAGCCCATAG
• 3′ sequencing primer: tcgactCCTGCAGGaggaaac

Resource Information

• Supplemental Documents:
  • pdr111_mannose_m2operon_restrict_sbfi.gb

Terms and Licenses

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

Depositor Comments

Please note: Plasmid contains an early termination at W490 in Klf4. The binding and functional sites in the Klf4 protein are upstream of this, but it is not known how this early termination affects plasmid function.

The functional B. subtilis construct can be acquired from the corresponding author of the manuscript upon request