EF12J
(Plasmid #42943)

• 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: EN+ 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: AgeI-BclI fragment swapped with nt 1927-3708 from JM102 to create a hybrid L1RP/L1.3 element (L1.3 differs from L1RP by 10 base changes in the swapped region).
• GenBank ID: M80343.1
• Entrez Gene: L1RE1 (a.k.a. L1.2, LRE1)
• Promoter: CMV MIE, L1 5'UTR
• Tag / Fusion Protein:
  • EGFP (opposite strand) (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: pCEP_fwd; LNCX
• 3′ sequencing primer: EGFP-C

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.

I->T substitution at the end of ORF 2 should not affect plasmid function.