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OG19 - pSF-CMV-RSV-CAT 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.

Product Code: OG19

Size (bp): 5450

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 LTR promoter.

Purpose: This vector contains a Chloramphenicol Acetyl Transferase (CAT) reporter gene cassette that is designed as an add-on to any of our vectors. The RSV CAT cassette is flanked by AscI sites. The promoter and gene are not designed to be split up in this vector. If you would like the CAT gene alone, or the RSV promoter alone, please see the reporter gene section, or the promoter section, on our website. The RSV promoter is considerably weaker than CMV which can make low abundance CAT signals difficult to detect. We have a stronger Ubiquitin promoter driven vector in the same format if you require higher levels of reporter gene activity.

Quality Validation: This vector has been demonstrated to express genes to high levels under the CMV promoter when inserted into the main MCS. 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. Expression of CAT is simultaneously controlled by the RSV promoter, and this has been validated in the same cell line, but the expression level is relatively low in comparison to our ubiquitin and CMV driven CAT vectors.

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.

Inserting a Gene: Each restriction site in this vector has a purpose and allows the insertion of specific pre-designed DNA sequences. To insert a gene, we recommend using any sites between (and including) the HindIII and XbaI sites. By doing this you will be able to insert most of our other products upstream, or downstream, of your gene. Click here to read more about the purpose of each restriction site.

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 SnapFastTM system and to retain a high level of antibiotic resistance. It has been validated in E.coli.
  • The CAT gene has been modified to remove an NcoI site and an EcoRI site to ensure compatibility with all of our products. These changes were conservative and did not alter the amino acid sequence of the CAT protein.
  • 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 CAT 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.