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Zeocin Selection Plasmids

OG23 - pSF-CMV-Ub-Zeo 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-Ub Zeo AscI

Product Code: 
OG23

Quantity Provided:
 5µg

Size (bp): 
5927

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 Human Ubiquitin promoter

Purpose: This vector contains a Zeocin (Zeo) resistance expression cassette under control of the Ubiquitin (Ub) promoter that is designed as an add-on to any of our vectors. The entire Ub Zeo cassette is flanked by AscI site. This vector can be used to force recombination into the genome of mammalian cells following transfection and selection, although this method can be inefficient. It can also be used conjunction with the SV40 origin of replication in cells expressing the SV40 large T antigen (such as 293T cells) to make stable cells lines following transfection and selection.

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 was validated in the A549 lung carcinoma cell line. The Ub Zeocin cassette has also been validated in the same cell line and shown to induce resistance to Zeocin. 

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.

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 Zeocin gene has been modified to remove BseRI, FseI, SmaI, EagI and ApaLI sites to ensure compatibility with all of our products. These are all conservative changes and do not change the amino acid sequence.  
  • The Ubiquitin (Ub) promoter in this vector has been modified to remove BseRI, StuI and SpeI sites. These changes do 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, Ubiquitin Zeocin 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.