pMT870 human L1 RT- (ORF2p D702Y) ORF2p-3xFlag (ORFeus-Hs, CMV promoter) in pCEP4 Puro (derivative of pMT646)
(Plasmid
#213027)
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PurposeExpresses reverse transcriptase dead (RT- ORF2p D702Y) full length human LINE-1 in human cells (codon optimized ORFeus-Hs), with a C-terminal 3xFlag tag on ORF2
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 213027 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepCEP4 Puro
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Backbone manufacturerInvitrogen (modified)
- Backbone size w/o insert (bp) 9879
- Total vector size (bp) 15135
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Modifications to backboneHygro replaced with Puro
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Vector typeMammalian Expression
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Selectable markersPuromycin
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert nameHuman LINE-1 (ORFeus-Hs codon optimized sequence, RT- D702Y) with ORF2-3xFlag
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Alt nameRT- LINE-1
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SpeciesH. sapiens (human)
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Insert Size (bp)5256
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MutationD702Y (RT- reverse transcriptase catalytic dead)
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GenBank IDAAC51271.1
- Promoter CMV
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Tag
/ Fusion Protein
- 3xFlag (ORF2p) (C terminal on insert)
Cloning Information
- Cloning method Gibson Cloning
- 5′ sequencing primer CMV_Forward
- 3′ sequencing primer T7 Term (Common Sequencing Primers)
Resource Information
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Supplemental Documents
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
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)
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
pMT870 human L1 RT- (ORF2p D702Y) ORF2p-3xFlag (ORFeus-Hs, CMV promoter) in pCEP4 Puro (derivative of pMT646) was a gift from Martin S. Taylor (Addgene plasmid # 213027 ; http://n2t.net/addgene:213027 ; RRID:Addgene_213027) -
For your References section:
Structures, functions and adaptations of the human LINE-1 ORF2 protein. Baldwin ET, van Eeuwen T, Hoyos D, Zalevsky A, Tchesnokov EP, Sanchez R, Miller BD, Di Stefano LH, Ruiz FX, Hancock M, Isik E, Mendez-Dorantes C, Walpole T, Nichols C, Wan P, Riento K, Halls-Kass R, Augustin M, Lammens A, Jestel A, Upla P, Xibinaku K, Congreve S, Hennink M, Rogala KB, Schneider AM, Fairman JE, Christensen SM, Desrosiers B, Bisacchi GS, Saunders OL, Hafeez N, Miao W, Kapeller R, Zaller DM, Sali A, Weichenrieder O, Burns KH, Gotte M, Rout MP, Arnold E, Greenbaum BD, Romero DL, LaCava J, Taylor MS. Nature. 2024 Feb;626(7997):194-206. doi: 10.1038/s41586-023-06947-z. Epub 2023 Dec 14. 10.1038/s41586-023-06947-z PubMed 38096902