The pre-linearized template vector contains the T7 promoter, transcription initiation sequence (AGG), 5′-UTR (untranslated region), 3′-UTR, and poly(A) sequence (Figure 1A and Figure 2).

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 1B). Download sequence information for the Linearized Template Vector (Hind III) or Linearized Template Vector (BspQ I) in fasta formats. 

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 and BspQ I are recommended to linearize an IVT template when using our template vectors, HindIII and BspQ I vectors respectively (as 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.

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. Download sequence information for the Fluc control fragment (fasta format).

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 or Takara IVTpro mRNA Synthesis System for a step-by-step procedure.