EF05J
(Plasmid #42941)

• Depositing Lab: Eline Luning Prak
• Publication: Farkash et al Nucleic Acids Res. 2006 Feb 28;34(4):1196-204. Print 2006.

Backbone

• Vector backbone: pCEP4
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 10410
• Total vector size (bp): 23135
• Modifications to backbone: Blunt ligation of nt 1-1392 of pPur (BD/Clontech) into the NruI site of pCEP4 to insert PuroR
• Vector type: Mammalian Expression
• Selectable markers: Puromycin, Hygromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Negative control Functionally Dead Human L1 element
• Alt name: LINE-1
• Alt name: Long Interspersed Element 1
• Alt name: L1RT
• Species: H. sapiens (human)
• Insert Size (bp): 7904
• Mutation: ORF1 R261A, R262A
• GenBank ID: M80343.1
• Entrez Gene: L1RE1 (a.k.a. L1.2, LRE1)
• Tag / Fusion Protein:
  • EGFP (opposite strand) (C terminal on insert)

Terms and Licenses

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

Depositor Comments

These plasmids consist of the L1RP element tagged with an enhanced green fluorescent protein (EGFP) cassette (pL1RP-EGFP) in a pCEP4 backbone (Invitrogen). The L1 element is driven by its 5′-UTR and an upstream CMV MIE promoter. L1-EGFP (EF06R, Addgene plasmid #42940) was derived from L1RP-EGFP by blunt ligation of 1–1392 nt of pPur (BD/Clontech) into the NruI site of pCEP4.

A negative control ‘dead’ L1-EGFP (EF05J, Addgene plasmid #42941) was similarly derived from pL1RP(JM111)-EGFP, which contains disabling mutations in ORF1.

An EN− plasmid (EF13E, Addgene plasmid #42942) was created by swapping 1927–3708 nt (Age I–Bcl I) from JM102D205A, which contains an endonuclease function-abrogating D205A point mutation in the EN domain, into EF06R.

A control plasmid (EF12J, Addgene plasmid #42943) was similarly derived from JM102. JM102 and JM102D205A are derived from L1.3, which differs from L1RP by 10 base changes in the region swapped.

The A250V mutation found during Addgene QC is conservative and should not affect function.