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SEAP

OG17 - pSF-CMV-RSV-SEAP Ascl

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Full Plasmid Details (PDF)

Sequence (txt)

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: OG17

Size (bp): 6356

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 plasmid contains a Secreted Alkaline Phosphatase (SEAP) cassette that is designed as an add-on to any of our other plasmids. The RSV SEAP cassette is flanked by AscI sites but is not designed to be split up. If you would like the SEAP 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 SEAP signals difficult to detect. We have a stronger Ubiquitin promoter driven plasmid in the same format if you require higher levels of reporter gene activity.

Transcription Termination: This plasmid contains three alternative transcription terminators for mammalian, bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

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.

Quality Validation: This plasmid has been demonstrated to express reporter genes, other than SEAP, 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 RSV SEAP cassette has also been validated in the same cell line.  

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 SEAP gene has been modified to remove SalI, Hind3, SphI, BamHI, NcoI, NdeI, Avr2, Sac2, NcoI, BspHI, StuI, EagI sites and two SacI sites, two NarI sites, two SmaI sites, as well as three BseRI sites and six BsgI sites to ensure compatibility with all of our products.
  • The Rous Sarcoma Virus (RSV) promoter in this plasmid 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 plasmid at two sites positioned to flank the promoter, start codon, origin, RSV SEAP 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.