pMT1093 human L1 EN- (ORF2p E43S, D145N) ORF2p-3xFlag (ORFeus-Hs, CMV promoter) in pCEP4 Puro (derivative of pMT646)
(Plasmid #213028)

• Purpose: Expresses endonuclease dead (EN- ORF2p E43S, D145N) full length human LINE-1 in human cells (codon optimized ORFeus-Hs), with a C-terminal 3xFlag tag on ORF2
• Depositing Lab: Martin S. Taylor
• Publication: Structures, Functions, and Adaptations of the Human LINE-1 ORF2 Protein (unpublished)

Backbone

• Vector backbone: pCEP4 Puro
• Backbone manufacturer: Invitrogen (modified)
• Backbone size w/o insert (bp): 9879
• Total vector size (bp): 15135
• Modifications to backbone: Hygro replaced with Puro
• Vector type: Mammalian Expression
• 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: Human LINE-1 (ORFeus-Hs codon optimized sequence, EN- E43S D145N) with ORF2-3xFlag
• Alt name: EN- LINE-1
• Species: H. sapiens (human)
• Mutation: E43S D145N double mutant EN- endonuclease catalytic dead
• GenBank ID: AAC51271.1
• Promoter: CMV
• Tag / Fusion Protein:
  • 3xFlag (ORF2p) (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CMV_Forward
• 3′ sequencing primer: SV40pA-Rev

Resource Information

• Supplemental Documents:
  • pMT1093 ORFeus CMV-L1 ORF2-3xFlag EN- E43S D145N.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• 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).