Vector Cloning
As the world’s leading provider of custom vector cloning services, VectorBuilder can create any custom DNA vector to meet your research needs. Our platform features about 200 optimized vector systems, validated through in vitro and in vivo (when applicable) testing. Outsourcing your cloning projects to VectorBuilder is often more cost-effective than in-house cloning in terms of reagent and labor cost, with the added benefit of saving your time, effort, and the frustration associated with cloning.
In addition to molecular cloning of vectors, we offer several related services including plasmid DNA preparation, virus packaging, library construction, stable cell line engineering, and therapeutic IVT RNA synthesis.
Highlights
Free, highly-intuitive platform for quick and easy design of custom vectors using our vast inventory of functionally validated vector backbones and components.
Modular “Lego-like” production workflow enabling parallel cloning of custom vectors efficiently and reliably, with over 95% of projects completed on time.
Extensive experience in cloning complex and challenging vectors.
Full ownership of the IP for your custom vectors and free vector storage.
Receive experiment-ready vectors delivered to your bench
Order Now!Service Information
Overview and workflow of custom cloning services
*Price depends on the type of sourcing, backbone, and cloning type.
Price and turnaround Price Match
While >80% of the vector components can be directly sourced from our in-house collections (based on past production data), we can create vectors from scratch (de novo synthesis) or from customer-supplied materials (vector backbone or gene template). Our custom vector cloning workflow includes QC of customer-supplied materials (if applicable), sourcing of vector components and vector backbones, the actual vector cloning, and final vector QC. A brief overview of price and turnaround for our custom cloning services are shown in Table 1 and 2:
Table 1. Simple vector cloning*
| Vector Type | Price (USD) | Turnaround |
|---|---|---|
| shRNA vector | $149 | 4-7 days |
| gRNA vector (single-gRNA) | $149 | 4-7 days |
| gRNA vector (dual-gRNA) | $329 | 6-12 days |
| Expression vector | $139 | 3-6 days |
Table 2. De novo synthesis
| Fragment Length | Price (USD)** | Turnaround |
|---|---|---|
| <1.5 kb | $0.12/bp | 5-10 days |
| 1.5-3 kb | $0.14/bp | 10-15 days |
| 3-5 kb | $0.17/bp | 10-20 days |
| 5-8 kb | $0.21/bp |
*Simple vector refers to vectors that can be constructed with 1-step cloning using VectorBuilder's popular in-stock backbones and components.
**The de novo gene synthesis fee may be higher when 1) the fragment contains regions that are difficult to synthesize such as high GC content, simple repeats or segmental repeats; 2) fewer than three DNA fragments are synthesized.
Technical Information
Quality control (QC)
All vectors cloned by VectorBuilder come with a 100% sequence accuracy guarantee. The table below lists our QC items for custom vector cloning:
| Raw Material | ||
|---|---|---|
| Vector backbone | Vector components | |
| VectorBuilder's in-stock material |
|
|
| Customer-supplied |
|
|
| De novo synthesized | - |
|
| Final Vector | ||
|
||
Customer-supplied materials
If customer-supplied materials such as backbones or gene templates are needed, please submit your materials information on "Support" > "Materials Submission Form". Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur an additional QC charge starting from $100 per item, depending on the type and usage of item. Please note that production cannot be initiated until customer-supplied materials pass QC.
Resources
FAQ
At VectorBuilder, a combination of various molecular biology techniques is utilized to generate custom vectors based on customer needs. While expression vectors are typically constructed using Gateway cloning or Gibson assembly, shRNA and gRNA vectors are cloned using a ligation-based approach to provide you with your desired vectors at the lowest prices and fastest turnaround times utilizing our highly optimized high-throughput cloning platform. Additionally, we routinely use a variety of other methods including de novo gene synthesis and Golden Gate cloning as needed, on a project-by-project basis.
All vectors at VectorBuilder are cloned using our proprietary host strain VB UltraStable™, which is designed to achieve high transformation efficiency (>1x109 cfu/ug) and for propagating DNA plasmids with unstable elements such as repeated sequences. Therefore, we highly recommend that you use VB UltraStable™ chemically competent cells for propagating your plasmids obtained from VectorBuilder. However, our vectors are compatible with and can be propagated in other commonly used bacterial hosts strains such as DH5α and Stbl3.
The origin of replication on your plasmid is for low-copy replication
The number of plasmid copies per bacterial cell is determined by the origin of replication on the plasmid. Some origins have inherent low copy number. Check the copy number of the origin of replication on your plasmid. For low-copy plasmids, increase the amount of E. coli culture for plasmid DNA prep in order to obtain satisfying DNA yield.
Check the copy number of the origin of replication of plasmids supplied by VectorBuilder
The volume of bacterial culture is too low for plasmid prep column
Please check the binding capacity of your plasmid prep column and whether your plasmid is high- or low-copy plasmid. For mini preps, we recommend that you harvest 1-5 ml of overnight bacterial culture. For maxi preps, if the plasmid is high-copy plasmid, we recommend using 100-150 ml of overnight bacterial culture; if the plasmid is low-copy plasmid, we recommend using 300-500 ml of overnight bacterial culture. Typically, for high-copy plasmid, ~5 ug of plasmid DNA can be extracted from every 1 ml of culture in mini prep and ~500 ug of plasmid DNA can be obtained from 150 ml of culture; for low-copy plasmid (e.g. pET), 1.5-2.5 ug of plasmid DNA can be harvested from every 1 ml of culture in mini prep and 150-200 ug of plasmid DNA can be obtained from 150 ml of culture.
Only a fraction of bacteria in the liquid culture contain plasmids
Some antibiotics, ampicillin in particular, degrade fast in liquid culture. As a result, bacteria that do not contain plasmids can propagate to a significant fraction of the culture, causing poor yield of the plasmid prep. To avoid this, please prepare ampicillin containing growth medium freshly before use and make sure that enough ampicillin is supplied. Also, when culturing ampicillin-resistant bacteria, do not let the liquid culture saturate for too long before harvesting. Besides insufficient antibiotics in the culture, extracting plasmid DNA from very old culture can also result in low yield, since many bacterial cells are dead and plasmid DNA they contain is degraded. Therefore, try to extract plasmid DNA from fresh culture. If plasmid prep cannot be performed immediately, you can spin down the bacteria and store the pellet in -80°C freezer for later plasmid prep.
The liquid culture is directly inoculated from E. coli stab culture
Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. Detailed user instructions can be found by going to menu item Learning Center > Documentation > Stab Culture.
You have not carefully followed the manual of the plasmid prep kit
If you use a plasmid prep kit, please carefully read the manual before use. Improper operations can often lead to poor performance of the kit.
The mini/maxi prep column is low-quality
Some brands of plasmid DNA prep columns perform poorly or inconsistently for DNA preparation.
If our QC assays uncover a synonymous mutation, we will contact you for approval in moving forward with cloning, as this mutation will be unlikely to affect experimental outcome. However, if a non-synonymous mutation is found, we will discuss with you the best course of action for correction before cloning and downstream services are initiated.
Documents
User Instructions Brochures & Flyers Material Safety Data Sheet (MSDS) Certificate of Analysis (COA)Featured Citations
Large language models generate functional protein sequences across diverse families
Ali Madani, et al.
Nature Biotechnology, 2023, doi: 10.1038/s41587-022-01618-2
Products used:
Abstract: Generating artificial protein sequences using artificial intelligence could enable breakthrough solutions for biomedical and environmental challenges. Viewing amino acid sequences as a language, we demonstrate that a deep learning-based language model can generate functional artificial protein sequences across large protein families, akin to generating grammatically and semantically correct natural language sentences on diverse topics. Our protein language model is trained by simply learning to predict the next amino acid for over 280 million protein sequences from thousands of protein families, without explicit biophysical modeling. We experimentally evaluate model-generated artificial proteins fine-tuned to five distinct antibacterial lysozyme families. Artificial proteins show similar activities and catalytic efficiencies as representative natural lysozymes, including hen egg white lysozyme, while maintaining activity with as low as 31.4% identity to any known naturally-evolved protein. The X-ray crystal structure of an enzymatically active artificial protein recapitulates the conserved fold and positioning of active site residues found in natural proteins. We show our language model’s ability to be adapted to different protein families by accurately predicting the functionality of artificial chorismate mutase and malate dehydrogenase proteins. These results indicate that neural language models successfully perform artificial protein generation across protein families and may prove to be a tool to shortcut evolution.
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Surficial nano-deposition locoregionally yielding bactericidal super CAR-macrophages expedites periprosthetic osseointegration
Ziyang Li, et al.
Science Advances, 2023, doi: 10.1126/sciadv.adg3365
Products used:
Abstract: Tracking and eradicating Staphylococcus aureus in the periprosthetic microenvironment are critical for preventing periprosthetic joint infection (PJI), yet effective strategies remain elusive. Here, we report an implant nanoparticle coating that locoregionally yields bactericidal super chimeric antigen receptor macrophages (CAR-MΦs) to prevent PJI. We demonstrate that the plasmid-laden nanoparticle from the coating can introduce S. aureus-targeted CAR genes and caspase-11 short hairpin RNA (CASP11 shRNA) into macrophage nuclei to generate super CAR-MΦs in mouse models. CASP11 shRNA allowed mitochondria to be recruited around phagosomes containing phagocytosed bacteria to deliver mitochondria-generated bactericidal reactive oxygen species. These super CAR-MΦs targeted and eradicated S. aureus and conferred robust bactericidal immunologic activity at the bone-implant interface. Furthermore, the coating biodegradability precisely matched the bone regeneration process, achieving satisfactory osteogenesis. Overall, our work establishes a locoregional treatment strategy for priming macrophage-specific bactericidal immunity with broad application in patients suffering from multidrug-resistant bacterial infection.
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Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease
Anna B Meier, et al.
Nature Biotechnology, 2023, doi: 10.1038/s41587-023-01718-7
Products used:
Abstract:The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.
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