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Puromycin Selection Plasmids

OG21 - pSF-CMV-Ub-Puro 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: OG21

Size (bp): 6152

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 plasmid contains a Puromycin (Puro) resistance expression cassette under the transcriptional control of the Ubiquitin (Ub) promoter that is designed as an add-on to any of our other plasmids. The Ub Puro cassette is flanked by AscI sites but is not designed to be split up. If you would like the Puromycin gene alone, or the Ub promoter alone, please see the mammalian antibiotic selection gene section, or the promoter section, on our website. This plasmid 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 cell lines following transfection and selection.

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.

Quality Validation: This plasmid 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 the A549 lung carcinoma cell line. The Ub Puro cassette has also been validated in the same cell line and shown to induce resistance to Puromycin. 

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 Puromycin gene has been modified to remove a StuI site and an EagI site to ensure compatibility with all of our products. These are all conservative changes and do not change the amino acid sequence.
  • The Ubiqitin (Ub) promoter in this plasmid 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 plasmid at two sites positioned to flank the promoter, start codon, origin, Ubiquitin Puromycin 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.