Plasmid Vector Assembly
Plasmid Vector Assembly - Related Videos
07:37
CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion
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2017
Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.
06:25
Use of In Vivo Assembly for High-efficiency Plasmid Construction
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Cited by 2 •
2025
In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.
09:51
Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly
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Cited by 2 •
2016
This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
10:18
Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
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Cited by 8 •
2015
Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.
FAQs
Sequence compatibility determines how DNA fragments join and whether the final molecule preserves the intended arrangement. The plasmid backbone provides the circular framework, while compatible junctions allow inserts to connect in a specified order and orientation. This matters because an incorrectly oriented or incomplete insert can alter downstream gene expression or prevent the construct from performing its planned function.
These elements determine how the construct behaves after introduction into a host cell. Promoters help control when and where an inserted gene is expressed, selectable markers support identification of cells carrying the plasmid, and regulatory sequences provide additional control over gene activity. Choosing compatible elements helps align the plasmid design with its intended biological function.
Insert orientation determines how the sequence relates to nearby promoters and regulatory elements. A correctly oriented insert can support the intended direction of transcription and expression, whereas reversed placement may disrupt the planned activity of the construct. Orientation therefore becomes especially important in expression studies, reporter assays, recombinant protein production, and other experiments requiring controlled gene activity.
The assembly design can place one or more inserts within a single plasmid backbone in a planned order. Sequence-compatible junctions help connect the fragments into one continuous molecule while preserving their intended relationships. This arrangement allows researchers to combine coding sequences with promoters, regulatory elements, or other selected components when a study requires coordinated functions within the same construct.
A typical workflow begins by designing the backbone, inserts, promoters, selectable markers, and regulatory sequences for the intended function. Researchers then join the selected DNA components using a sequence-compatible assembly approach, introduce the resulting plasmid into host cells, and verify the construct. Each stage connects design choices with confirmation that the assembled molecule matches expectations.
Assembled plasmids support several experimental goals, including gene cloning, recombinant protein production, reporter assays, genome editing, and functional studies. The appropriate design depends on whether researchers need replication, controlled expression, or another plasmid-based function in host cells. Verification is important because the biological interpretation of these experiments depends on the construct performing as intended.