pRT006.3 L1 dual-luciferase AI retrotransposition reporter
(Plasmid #213031)

• Purpose: Bi-directional luciferase antisense intron (firefly fluc AI) human LINE-1 retrotransposition reporter (ORFeus-Hs sequence) for sleeping beauty integration
• Depositing Labs: Donna Romero, Martin S. Taylor
• Publication: Structures, Functions, and Adaptations of the Human LINE-1 ORF2 Protein (unpublished)

Backbone

• Vector backbone: pCEP Puro
• Backbone manufacturer: Genscript / Invitrogen
• Backbone size w/o insert (bp): 8859
• Total vector size (bp): 18643
• Vector type: Mammalian Expression, Luciferase
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Tet-On Human LINE-1 (ORFeus-Hs) Firefly + Renilla Luciferase Antisense Intron dual retrotransposition marker
• Species: H. sapiens (human)
• Insert Size (bp): 9784
• Promoter: pTRE bidirectional tet-on
• Tag / Fusion Protein:
  • Firefly luciferase antisense intron (3' UTR) (C terminal on backbone)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: bidir_pTRE_Fwd (CGCCACCTCGACATACTCG)
• 3′ sequencing primer: SV40pA-Rev

Resource Information

• Supplemental Documents:
  • prt006.3.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
  • Renilla Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The construct is based on the synthetic L1 ORFeus-Hs sequence generated in the lab of Jef Boeke (see W. An et al., Mobile DNA 2, 2011 https://doi.org/10.1186/1759-8753-2-2)

For integration with Sleeping Beauty such as SB100x (Izsvak lab, Addgene #34879). This vector is similar to pYX056 from An lab, see also:
Xie, Y. et al. Cell division promotes efficient retrotransposition in a stable L1 reporter cell line. Mob DNA 4, 10 (2013). https://doi.org/10.1186/1759-8753-4-10
and
Xie, Y., Rosser, J. M., Thompson, T. L., Boeke, J. D. & An, W. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. Nucleic Acids Res 39, e16 (2011). https://doi.org/10.1093/nar/gkq1076