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Cloning Kit for mRNA Template

Cloning Kit for mRNA Template (Cat. # 6143) is a unique In-Fusion Cloning-based system used to prepare a template for the IVT reaction. The kit comes with an In-Fusion-ready, linearized T7 template vector into which the coding sequence (CDS) of your gene of interest (GOI) can be seamlessly cloned.

Components
Linearized Template Vector (50 ng/μl)
FLuc Control Fragment (100 ng/μl)
5X In-Fusion Snap Assembly Master Mix

Linearized Template Vector  

The pre-linearized template vector contains the T7 promoter, transcription initiation sequence (AGG), 5′-UTR (untranslated region), 3′-UTR, and poly(A) sequence (Figure 1).

Figure 1. Linearized Template Vector (2,921 bp) in Cloning Kit for mRNA Template.

Because the Linearized Template Vector has the transcription start site “AGG” as a default sequence, it is compatible with CleanCap Reagent AG (TriLink BioTechnologies, No. N-7113 or N-7413, not included). This allows for convenient, co-transcriptional capping.

The Linearized Template Vector also has 15 bp of In-Fusion cloning sites at the end of 5′-UTR and at the beginning of 3′-UTR, where your GOI can be inserted (Figure 2). Download sequence information for the Linearized Template Vector (fasta format). 

Figure 2. A detailed view of Linearized Template Vector. The T7 promoter, transcription initiation sequence (AGG), 5'-UTR (untranslated region), In-Fusion cloning sites, 3'-UTR, and poly(A) sequence are shown.

Insert design and preparation  

Begin by designing the coding sequence. In order to optimize codon composition in the coding region, we recommend using commercially available codon optimization tools. Once you determine the coding sequence, make sure your insert has the features below.

  1. Start and stop codons—even though the vector has a stop codon, a CDS containing the stop codon must be prepared to ensure that translation is completed at the desired location.
  2. Absence of restriction site(s) recognized by a linearizing enzyme—make sure your insert does not have a restriction site for the enzyme used for linearization. HindIII is recommended to linearize an IVT template when using our linearized template vector (as is shown in Figure 2).
  3. In-Fusion cloning sites—these 15 bp sequences homologous to those in the Linearized Template Vector should be included. If you are generating the In-Fusion cloning sites within the insert using PCR, refer to the example primer design shown in Figure 3.

Figure 3. Example PCR primer design for a FLuc CDS fragment. The 15-base sequence for In-Fusion Cloning is shown in red, the start codon (ATG) in blue, and the stop codon (TGA: complementary strand) in green.

FLuc Control Fragment  

The FLuc Control Fragment (1,683 bp) included in this cloning kit can be used as an assay control for In-Fusion Cloning. The fragment contains the 3′ and 5′ In-Fusion sequences (15 bp) and the CDS of firefly (Photinus pyralis) luciferase, optimized for expression in human cells (Figure 4). The sequence is identical to base pairs 2,685–4,367 (1,683 bp) of the Positive Control Template (FLuc) included with the Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144). Download sequence information for the Fluc control fragment (fasta format).



Figure 4. FLuc Control Fragment (1,683 bp). The FLuc coding sequence (gray box) spans a start codon (ATG) and a stop codon (TGA).

5X In-Fusion Snap Assembly Master Mix  

This master mix enables directional and seamless cloning of any fragments into any vector without sequence constraints. Its exceptionally high accuracy makes it ideal for preparing the DNA template for in vitro transcription. It is the same component included in the In-Fusion Snap Assembly Master Mix (Cat. # 638947).

If you use PCR amplification to prepare your insert, the PCR product should be purified and quantified before In-Fusion Cloning. Please see the User Manual for Cloning Kit for mRNA Template (Cat. # 6143) or Takara IVTpro mRNA Synthesis System (Cat. # 6141) for a step-by-step procedure.

Related Products

Cat. # Product Size License Quantity Details
6141 Takara IVTpro™ mRNA Synthesis System 1 Set USD $398.00

Takara IVTpro mRNA Synthesis System includes all components necessary from DNA template preparation to high-yield in vitro transcription reaction. Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield. This kit includes the Cloning Kit for mRNA Template (Cat. # 6143) and Takara IVTpro mRNA Synthesis Kit (Cat. # 6144), which are also sold separately.

Notice to purchaser

This product is for in vitro diagnostic use. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from Takara Bio Inc. If you require licenses for other use, please contact us by phone at +81 77 .565. 6976 or from our website at https://www.takara-bio.co.jp/inquiry/ivd/. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.

Documents Components Image Data
6143 Cloning Kit for mRNA Template 10 Rxns USD $179.00

Cloning Kit for mRNA Template enables easy cloning of a template DNA into a pre-linearized plasmid (included in the kit) for downstream in vitro transcription reactions. The pre-linearized plasmid contains the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs (untranslated region), and poly(A) sequence. The kit also includes an In-Fusion Snap Assembly Master Mix for seamless cloning of the target DNA into the pre-linearized vector and a FLuc Control Fragment, consisting of 15 bp 3' and 5' In-Fusion sequences and Fluc CDS (1,683 bp).

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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6144 Takara IVTpro™ T7 mRNA Synthesis Kit 20 Rxns USD $248.00

Takara IVTpro mRNA Synthesis Kit enables the production of large amounts of mRNA through in vitro transcription using an optimized, highly efficient T7 RNA polymerase. The kit includes separate NTPs for easy optimization or replacement with modified nucleosides, such as pseudouridine. The kit synthesizes up to 200 μg or more of mRNA per 20 μl reaction, includes DNase I to digest the template DNA after the in vitro transcription reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA for downstream applications. 

The kit also includes a Positive Control Template (FLuc) plasmid containing the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs, FLuc CDS, and poly(A) sequence.

Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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