Antibiotic-Free RNA-OUT Marker for Mammalian Expression and DNA Vaccine Vectors, (manual)
• Replaces KanR resistance gene with antisense RNA-OUT, 150bp RNA marker
• Kit includes strain for selection with RNA-OUT
• Enhanced expression with optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)
• Complies with regulatory guidance
• Increased vaccine potency and safety by replacement of KanR gene
• Improved DNA backbone for maximum plasmid production in E. coli (>2.5g/L)
Description
Antibiotic resistance markers, typically kanamycin resistance (KanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. To ensure safety, however, regulatory agencies recommend elimination of antibiotic-resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to the potential transfer of antibiotic resistance to endogenous microbial flora and the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome.
Now, NTC has designed an antibiotic-free selection system (Fig. 1). The KanR gene has been replaced with a 150bp RNA-OUT antisense RNA, which represses expression of a counter selectable marker (SacB) from the host chromosome.
NTC offers two of it's popular mammalian expression and immunization vectors (equivalent to NTC7482 and NTC7485), now in the antibiotic-free format:
NTC8485-eRNA41H-EGFP: native (no tag), reporter for use as a control, or removed in order to clone a new gene, with antibiotic-free RNA-OUT selection on sucrose
NTC8482-eRNA41H: secreted (TPA tag), empty, ready for cloning a new gene of interest
In addition, NTC offers a retrofitting service, directly replacing the Kan gene in your current vectors with the RNA-OUT cassette.
Fig. 1: RNA-OUT selectable marker
Fig 2. NTC8485-EGFP. Marker is removed to clone a gene of interest.
Vector Features: trpA prokaryotic terminator: 3739-28; Primosomal assembly site (PAS-BH) extended origin: 32-316; pUC replication origin: 317-1331; Sucrose selection marker (RNA-OUT): 1348-1492; SV40 enhancer: 1493-1719; CMV enhancer: 1726-2283; CMV promoter: 2284-2403; Untranslated leader (exon 1): 2404-2590; HTLV-1 R-U5: 2475-2700; Synthetic Rabbit β-globin-based 3’ intron: 2709-2815; Intron: 2591-2815; Exon 2 (SR-protein binding sites-Kozak):2816-2864; EGFP: 2865-3584; SalI-BglII cloning cassette: 2859-3594; Eukaryotic terminator: 3595-3729.
Antibiotic Free, Mammalian Expression and DNA Vaccine Vectors, NTC 8482, NTC 8485
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