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Addgene

pDual-eGFP
(Plasmid #63215)

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Full plasmid sequence is not available for this item.

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 63215 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pNL-eGFP/CEF
  • Backbone manufacturer
    Jakob Reiser
  • Backbone size w/o insert (bp) 10680
  • Total vector size (bp) 10947
  • Modifications to backbone
    A PCR product amplified from pET28a was ligated to the SmaI digested pNL-eGFP/CEF (Zhang et al. 2002; Zhang et al. 2004; Reiser et al. 2000) and screened for gain of green fluorescence using a Dark Reader light box. The PCR product starts with a BglII sequence and then amplifies the T7 promoter, the LacI binding site (Lac operator), a Shine-Dalgarno sequence, His6, T7 tag, and ends at the beginning of the pET28a MCS. eGFP was strongly expressed from the Rosetta-gami™2(DE3) strain, which bears the T7 RNA polymerase gene. If desired, the insert may be excised again by cleavage with BglII and BamHI and religating. NcoI/XhoI excises eGFP and produces a product that can by ligated to pET28a (NcoI/XhoI) to create a T7 expressed eGFP with a C-terminal His tag for purification.
  • Vector type
    Mammalian Expression, Bacterial Expression, Lentiviral

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    Stbl3
  • Growth instructions
    eGFP is strongly expressed in the Rosetta-gami™2(DE3) strain (EMD Millipore) following IPTG induction of the T7 RNA polymerase gene.
  • Copy number
    Unknown

Gene/Insert

  • Gene/Insert name
    His-T7-eGFP
  • Alt name
    His-tagged, T7-tagged enhanced green fluorescent protein
  • Species
    Synthetic
  • Insert Size (bp)
    864
  • GenBank ID
    KM019171
  • Promoter CMV-EF1α hybrid (CEF)
  • Tags / Fusion Proteins
    • His6 (N terminal on insert)
    • T7 (N terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site SmaI (destroyed during cloning)
  • 3′ cloning site SmaI (destroyed during cloning)
  • 5′ sequencing primer cgtcgccgtccagctcgaccag
  • 3′ sequencing primer gagcaagggcgaggagctgttc
  • (Common Sequencing Primers)

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Lentiviral vector with high transduction efficiency. Expression from the CMV-EF1α hybrid (CEF) promoter is very stable in mammalian cells. The lentivirus contains an SV40 origin 5’ to the eGFP gene. This influences gene targeting rates and the preferred strand for gene targeting and recombineering in cells expressing the SV40 T-antigen.

How to cite this plasmid ( Back to top)

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.

  • For your Materials & Methods section:

    pDual-eGFP was a gift from Richard S Myers (Addgene plasmid # 63215 ; http://n2t.net/addgene:63215 ; RRID:Addgene_63215)
  • For your References section:

    Fluorescent protein engineering by in vivo site-directed mutagenesis. Valledor M, Hu Q, Schiller P, Myers RS. IUBMB Life. 2012 Aug;64(8):684-9. doi: 10.1002/iub.1041. Epub 2012 May 28. 10.1002/iub.1041 PubMed 22639380