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pEGFP-puro
(Plasmid #45561)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 45561 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pEGFP-C1
  • Backbone manufacturer
    Clontech
  • Backbone size w/o insert (bp) 4731
  • Total vector size (bp) 5310
  • Vector type
    Mammalian Expression ; Bifunctional selection protein
  • Selectable markers
    Puromycin ; EGFP

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    Puromycin N-acetyl transferase
  • Alt name
    PuroR
  • Alt name
    EGFP-Puro
  • Insert Size (bp)
    600
  • Promoter Phcmv (Strong human cytomegalovirus immediate early promoter)
  • Tag / Fusion Protein
    • EGFP (N terminal on backbone)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site BglII (not destroyed)
  • 3′ cloning site BamHI (not destroyed)
  • 5′ sequencing primer EGFP-C; CMV-F
  • 3′ sequencing primer SV40-pA-R
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

Plasmid pEGFP-puro was constructed by PCR amplification of PuroR coding sequences from pPur (Clontech Laboratories, Palo Alto CA, USA) using upstream
(5'-AAGTCCGGACTCAGATCTAGGAGACGACCTTCCATGACCGAGT-3')
and downstream
(5'-GTTATCTAGATCCGGTGGATCCCGGGCACCGGGCTTGCGGGTCATGCACCA-3')
primers, digestion of the PCR product with BglII andBamHI, and ligation into BglII/BamHI digested pEGFP-C1 (Clontech Laboratories).

To construct a fusion protein containing both EGFP and PuroR sequences, PuroR coding sequences were ligated into an existing expression cassette for EGFP. The resulting fusion protein was designated EGFP-puro and is contained in plasmid pEGFP-puro. This plasmid contains the strong human cytomegalovirus immediate early promoter (Phcmv), the EGFP puro open reading frame, and the simian virus 40 (SV40) polyadenylation signal. The EGFP-puro protein contains the first 245 amino acids of EGFP. The C-terminal five amino acids of EGFP were deleted and replaced by four linker-encoded amino acids, followed by the complete 200-amino-acid PuroR sequence. The C-terminus is formed by 11 amino acids encoded by vector sequences. The resulting fusion protein (EGFP-puro) confers both green fluorescence and resistance to puromycin when expressed in mammalian cells.

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:

    pEGFP-puro was a gift from Michael McVoy (Addgene plasmid # 45561 ; http://n2t.net/addgene:45561 ; RRID:Addgene_45561)
  • For your References section:

    Bifunctional protein conferring enhanced green fluorescence and puromycin resistance. Abbate J, Lacayo JC, Prichard M, Pari G, McVoy MA. Biotechniques. 2001 Aug;31(2):336-40. 10.2144/01312st05 PubMed 11515370