pMT647 human L1 ORF2p-3xFlag only (ORFeus-Hs, CMV promoter, monocistronic construct) in pCEP4 Puro (derivative of pMT646)
(Plasmid #213029)

• Purpose: Expresses human LINE-1 ORF2 only 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• Publication: Structures, Functions, and Adaptations of the Human LINE-1 ORF2 Protein (unpublished)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): 9807
• Total vector size (bp): 14044
• Modifications to backbone: Hygro replaced with Puromycin resistance
• 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 ORF2p only (monocistronic, codon optimized ORFeus-Hs sequence) with ORF2-3xFlag
• Alt name: ORF2p
• Alt name: ORF2
• Alt name: LORF2_HUMAN
• Species: H. sapiens (human)
• Insert Size (bp): 4147
• GenBank ID: AAC51271.1
• Promoter: CMV
• Tag / Fusion Protein:
  • 3xFlag (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NotI (destroyed during cloning)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: CMV_Forward
• 3′ sequencing primer: SV40pA-Rev

Resource Information

• Supplemental Documents:
  • pMT647 human L1 ORF2p-3xFlag only (ORFeus-Hs, CMV promoter, monocistronic construct) in pCEP4 Puro.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).