GeneArt Elements Vector Construction
The GeneArt Elements parts collection comprises a growing subset of biologically well-characterized parts like promoters, terminators, enhancers, operators, open reading frames, etc., with exactly defined sequences and functionalities. Provided Elements parts can be combined with custom parts to design individualized vectors.
The GeneArt portal features an application that allows for the in vitro assembly of the parts. The intuitive drag-and-drop functionality of the portal facilitates the combination of parts on a symbolic level, without any limitations from the nucleic acid sequence at the junctions. The designed vector can be directly ordered via the portal. For further information regarding this service or the order process, please contact email@example.com
*Functionality in the final vector requires that you choose a resistance marker and an E. coli origin of replication from the GeneArt Elements repository
How to order
1). Order Online
Order online via our web portal
- Conveniently drag and drop GeneArt® Elements from the repository and add custom parts to create the vector of your choice.
- Directly order your custom-made vector online.
Your sequences will be synthesized as entered. However, please be aware that you need to pick a resistance marker and an E. coli origin of replication from the GeneArt Elements repository
The GeneArt Portal features an application which allows for the in vitro assembly of GeneArt Elements and custom parts. You can review relevant part information by clicking on the respective GeneArt Element in the repository (Figure 1). The information panel will showcase all part information (part name, functionality, sequence, etc.).
If you decide to include a GeneArt Element into your design simply drag and drop it into the construction panel.
In order to include a custom part please choose a generic part from the repository (e.g. generic promoter) and drag and drop it into the construction panel. Please fill in all required fields (e.g. part name, sequence information etc.).
The sequential order of parts in the construction panel will be the order of parts in the final construct. All parts will be seamlessly assembled, without leaving any scars. After finishing the design, vectors can be directly ordered via the portal.
Figure 1: Screen shot from the GeneArt Elements repository, exemplifying various DNA parts from the collection.
Application example (vector construction)
Some applications require very specific vectors that may not be commercially available without custom synthesis. In the example below, we created a set of vectors for efficient expression and secretion of a fluorescent protein to serve as a positive control for protein expression and secretion in mammalian cell lines (Figure 2). The vector may simultaneously serve as a control for the expression, secretion and purification of antibodies or His-tagged proteins.
|Construct||Replace this feature in Construct 1…||…with this feature to make the new construct|
||(Construct 1 is as shown)||(Construct 1 is as shown)
||TEV recognition site||Factor Xa recognition site|
||IgK secretion leader||tpa secretion leader|
||Neo without tags||Neo with 4 tags|
||Neo with stuffer between CDS and terminator||Neo without stuffer|
||CMV promoter||EF-1alpha promoter|
||CMV promoter||UbC promoter|
||IgK secretion leader||no secretion leader|
||emGFP-IgG1 Fc fusion protein||emGFP STOP|
Figure 2: Eleven vectors created using GeneArt Elements parts. Various DNA parts from the repository were used to construct 11 vectors that were designed to produce secreted fluorescent proteins, demonstrating the utility of the Elements parts collection.
Multiple vector combinations, coding for secreted green fluorescence protein (GFP), have been designed using parts from the repository.
As an example, two different types of protein secretion leaders were tested for GFP, the tissue plasminogen activator (tPA) secretion signal sequence and the immunoglobulin kappa secretory signal (IgK). Following transfection of the vectors into Freestyle293TM suspension cells, GFP fluorescence was visualized using UV light (Figure 3). In addition, microscopic images were taken using the FLoid® Cell Imaging Station (Figure 4). While cellular GFP displayed a homogenous distribution within the cells, the two secreted GFP variants showed a prominent vesicular staining typical for secretory pathway proteins. Moreover, conditioned media of cells expressing the indicated GFP variants were subjected to immunoblot analysis (Figure 5). Using a GFP-specific antibody, recombinant secreted GFP was nicely detected in cell culture supernatants with both CMV‑tPA leader+emGFP and CMV‑IgK leader+emGFP.
This shows that the GeneArt Elements vector constructions were functional, generating positive controls for protein secretion.
Figure 3: Direct visualization of GFP expression by fluorescence of the cell culture supernatant. Cell culture of mock and GFP transfected Freestyle™ 293 cells under UV light. Comparison of fluorescent intensity using different protein secretion leader signals.
GeneArt Elements Parts Repository is a fast and convenient tool for designing the appropriate vectors for your needs. We have constructed a variety of vectors that can be used as an expression and secretion control for various experimental setups. In this exemplary case, the secreted GFP protein is fused to the Fc part of the IgG1 antibody and a hexa-His-tag, thus it can be utilized as a positive control for Western blotting in combination with antibodies or His-tagged proteins.
The combinatorial assembly of different promoters, tags, secretion leader or antibiotic resistance cassettes can be exploited to find optimal part combinations for best performance, e.g. highest levels of protein expression and secretion.
Figure 4: Micrographs of transfected 293H cells. HEK 293 cells were transfected with the indicated constructs. Three days after transfection pictures were taken using a Floid Cell Imaging Station.
Figure 5: Western blot of pCMV-GFP vectors constructed with GeneArt Elements.
Frequently asked questions (FAQs)
Q. Are ordered constructs synthesized from scratch or do you re-use certain DNA Elements parts.
A. We would like to give our customers maximal flexibility in terms of their vector design. This requires in most instances the complete synthesis of the parts/vector.
Q. Is there any length limitation to the vector constructions offered?
A. Yes, the maximum length of a constructed vector is 12 kb.
Q. Are there any design restrictions?
A. No, all parts can be combined in all possible ways. However, vector functionality requires that you include at least one resistance marker and an origin of replication from the offered collection.
Q. Is it possible to optimize any open reading frames that I include?
A. Currently there is no optimization functionality within the vector construction application. However, ORFs can be optimized within the gene synthesis application of the GeneArt portal and then copied into the respective custom part.
Q. Is this the only way to order a vector construction within the portal?
A. No, you can also fill in the questionnaire/order form and send it to firstname.lastname@example.org