Creating Custom DNA Transposons for the TcBuster™ System
Molecular Guide
This guide is meant to be used as a resource to design DNA transposons (plasmids) for use with the TcBuster system. Bio-Techne does not guarantee the success of custom DNA transposons. It is the responsibility of the end user to design the transposon, assess the performance, and perform due diligence on any elements or sequences included in the construct design to determine freedom to operate.
Frequently Asked Questions
T cells, NK cells and other cell types can be affected by DNA-mediated toxicity when high concentrations of plasmid DNA are used. Minimizing the plasmid backbone by using a Nanoplasmid, or similar technology, allows for a higher ratio of GOI sequence to µg of plasmid delivered. The Nanoplasmid can be delivered to cells at a lower concentration than a traditional plasmid with the same gene editing efficiency. Improved viability and cell growth are observed after editing with lower concentrations of DNA.
Regulatory bodies, including the FDA, recommend that plasmids used in clinical applications do NOT contain antibiotic resistance genes for safety purposes. The Nanoplasmid does not contain a bacterial antibiotic resistance gene and is a good option for a TcBuster transposon.
Bio-Techne and Aldevron have partnered to streamline the ordering process for custom DNA transposons. Bio-Techne has banked a TcBuster backbone, fully equipped with TcBuster-compatible ITRs and a multiple cloning site, utilizing Aldevron’s Nanoplasmid vector system.
Once you have designed and built your custom DNA transposon insert, please contact Aldevron to perform gene synthesis, cloning, and manufacturing. We recommend a concentration of 2 mg/mL in nuclease free water. Email your request to nanoplasmids@aldevron.com.
The cost of ordering a transposon depends on the sequence complexity and size.
For initial custom Nanoplasmid construct production, a Nanoplasmid LULL and a one-time research license fee covering all preclinical work will be required.
The Nanoplasmid vector is licensed by Aldevron. You would need to negotiate your own license to manufacture the Nanoplasmid with another vendor.
If you are using a pUC backbone, or similar alternative, you will be able to perform the cloning and manufacturing in your own lab, or with a vendor of your choice.
Ordering Information
The TcBuster-compatible Nanoplasmid vector banked at Aldevron includes the following elements:
- TcBuster-compatible ITRs
- Human elongation factor 1 alpha (Ef1α) or MND promoter sequence
- Multiple cloning site (see step 2)
- Bovine growth hormone (BGH) poly(A) signal sequence
The backbone may be further customized by Aldevron to meet your needs.
You must provide the following elements (if applicable):
- Custom promoter sequence
- Kozak sequence
- Gene(s) of interest, separated by 2A elements if cargo is multicistronic
- Selection marker
- Custom poly(A) signal sequence
Build a linear map of the expression cassette in the molecular design software of choice, such as Snap Gene or Benchling.
Design Tips:
- Ef1α and MND are strong constitutive promoters suitable for mammalian overexpression.
- GCCACC is generally considered the classic consensus Kozak sequence and is the standard sequence used for TcBuster overexpression vectors.
- Typically, inserts will be cloned into the MluI/EcoRV sites, unless a custom promoter sequence is required.
- Self-cleaving peptides 2A element orientation: If the cargo is polycistronic, separate all genes from each other using GSG-2A elements.
- e.g., Gene 1-T2A-Gene 2-P2A-Gene 3-E2A-Gene 4-F2A.
- Typically, a different 2A element is used between each gene of a polycistronic construct. This avoids potential recombination issues during cloning.
- Data from a previous study suggests that the best 2A element order for optimal gene expression of a quad-cistronic construct is T2A-P2A-E2A.
Multiple cloning site with unique restriction enzyme sites in the TcBuster-compatible Nanoplasmid vector:
Confirm that the following elements are present and correct:
- The expression cassette is in the proper orientation: Promoter-Kozak-GOI-Poly(A) signal
- The correct promoter is used
- Kozak sequence immediately precedes the GOI
- GOI is in-frame and includes:
- Start codon
- Stop codon
- Properly placed GSG-2A elements, if applicable
- No premature transcriptional terminators (AATAAA or ATTAAA) are in the open reading frame
Run your expression cassette through the GenRCA Rare Codon Analysis Tool (optional).
- Analyze from start codon to stop codon.
- If codon adaptation index (CAI) is below 0.7, use GenSmart™ Codon Optimization or other equivalent codon optimization software to optimize heterologous expression of your GOI.
Contact us to perform gene synthesis, cloning, and manufacturing by filling out the form below.
Table 01 / Plasmid Map Elements
| Element | Sequence or Accession # | Region |
| TcBuster ITR upstream of cargo | Within plasmid map | 1371-1623 |
| Target site duplication | CTCTAGAG | Labeled TSD |
| EF1 alpha promoter | Sequence ID: PP944529.1 | 1677-2860 |
| BGH poly(A) signal | Sequence ID: MG437020.1 | 2971-3195 |
| TcBuster ITR downstream of cargo | Within plasmid map | 3332-3659 |
| Self-cleaving 2A peptides | https://www.nature.com/articles/s41598-017-02460-2 | See Nature publication |
Note: Region sequences are specific to the TcBuster pUC57 backbone with MCS plasmid sequence. The plasmid map of the TcBuster pUC57 backbone is available as a .dna file openable with programs such as SnapGene.
If you require further assistance, please email the technical support team at techsupport@bio-techne.com.
Receive your custom nanoplasmid design and quote today!
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