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Neomycin/G418/Geneticin Selection Plasmids

OG381 - pSF-CMV-RSV-Neo/G418 Ascl

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This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Vector: pSF-CMV-RSV-Neo/G418 AscI

Product Code: OG381

Quantity Provided: 5µg

Size (bp): 5585

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322   

Copy Number:  500-700 copies per cell

Promoter: Cytomegalovirus (CMV) immediate early promoter and the Rous Sarcoma Virus (RSV) LTR promoter

Purpose: This vector contains a Neomycin (Neo)/G418 resistance expression cassette under control of the RSV promoter. The entire RSV Neo/G418 cassette is flanked by AscI sites. The RSV promoter is considerably weaker than CMV. We have a stronger Ubiquitin promoter driven vector (pSF-CMV-Ub-Neo/G418 AscI) in the same format if you require high levels of reporter gene activity.  

G418 Selection notes: Although the gene name in this vector is neo, neomycin cannot be used in mammalian, or other eukaryotic cells, because it is ineffective at inhibiting eukaryotic ribosomes. For this reason, the plasmid must be selected in mammalian cells using G418 (otherwise known as Geneticin). The neo gene will induce resistance to kanamycin, neomycin and G418 in bacterial cells, however, in this vector the gene is not designed to express in bacteria, and hence will offer no resistance.  

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned within the XbaI site. Expression of reporters was validated in A549 cells. The RSV Neo/G418 cassette has also been validated in the same cell line and shown to induce resistance to G418. 

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The sequences and details of these primers are available through our website.

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

Genetic Modifications to standard parts:

  • CMV promoter – an NcoI site has been ablated in the CMV promoter. This change does not reduce expression.
  • KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.
  • The Neo/G418 gene has been modified to remove NcoI, EagI, PstI and SphI sites to ensure compatibility with all of our products. These are all conservative changes and do not change the amino acid sequence.   
  • The Rous Sarcoma Virus (RSV) promoter in this vector has been modified to remove an EcoRI site. This does not reduce expression.    

Restriction site notes:

  • Bgl2, KpnI, SwaI, AscI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, RSV Photinus luciferase cassette, and the KanR, respectively.
  • BsgI and BseRI recognise non-palindromic DNA sequences and cleave upstream of their recognition sites, within the stop codon that is found inside the XbaI restriction site.