hEF1α-H2B-mOrange2-IRES-EGFP PGK-Puromycin
(Plasmid #99277)

• Purpose: Dual fluorescent reporter - constitutive expression of mOrange2 (549/565) localized in the nucleus, EGFP (488/507) untargeted and visualized in the cytoplasm, and Puromycin resistance
• Depositing Lab: Jordan Miller
• Publication: Calderon et al Biomater Sci. 2017 Jun 29. doi: 10.1039/c7bm00223h.

Backbone

• Vector backbone: MillerLab H2B-mVenus-IRES-mPlum, DNA 2.0 Order #62961, Gene 179575
• Backbone manufacturer: DNA 2.0
• Modifications to backbone: cut out mVenus-IRES-mPlum with SalI and BamHI and insert mOrange2-IRES-EGFP
• Vector type: Lentiviral
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: H2B-mOrange2-IRES-EGFP
• Species: H. sapiens (human)
• Insert Size (bp): 2434
• Promoter: EF1 alpha-human
• Tags / Fusion Proteins:
  • mOrange2 (N terminal on insert)
  • H2B (N terminal on insert)
  • EGFP (N terminal on insert)
  • IRES (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: not sure
• 3′ sequencing primer: not sure

Gene/Insert 2

• Gene/Insert name: Puro
• Alt name: Puromycin
• Species: M. musculus (mouse)
• Insert Size (bp): 600
• Promoter: PGK-mouse
• Tag / Fusion Protein:
  • PGK (N terminal on backbone)

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: not sure
• 3′ sequencing primer: not sure

Resource Information

• Supplemental Documents:
  • H2B-mOrange2-IRES-EGFP.gb
  • Fluorescent Images
• A portion of this plasmid was derived from a plasmid made by: H2B-Venus sequence from Anna-Katerina Hadjantonakis: Addgene plasmid # 60141 (https://www.addgene.org/browse/sequence/60141/) Nowotschin, S., Xenopoulos, P., Schrode, N., & Hadjantonakis, A.-K. (2013). A bright single-cell resolution live imaging reporter of Notch signaling in the mouse. BMC Developmental Biology, 13, 15. doi:10.1186/1471-213X-13-15

Terms and Licenses

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