The comprehensive vector selection guide meticulously compiled by Synbio Technologies is to serve as a convenient reference tool for scientific researchers. This guide covers a wide range of vector types, ranging from classical plasmid vectors to cutting-edge viral vectors, as well as feature-rich cloning vectors and highly efficient expression vectors. We provide detailed information of each vector type, including the most important information such as its characteristics, advantages, limitations, and applications.

Vectors are DNA molecules used as a vehicle to carry target genes (DNA fragments ) into target cells, tissues or organisms so that the genes can be replicated and expressed. Vector’s ability to replicate in the host cell can prevent degradation and increase functional effectiveness. . Among all types of vectors, plasmid is the most commonly used in genetic engineering.

Plasmids are a class of nucleic acid molecules inherently present in biological cells that can replicate autonomously and be stably inherited independently of the host's chromosome, commonly found in prokaryotic bacteria and fungi. The vast majority of plasmids are DNA-based, with a minority being RNA-based. Natural DNA plasmids mostly possess a covalent, closed, and circular molecular structure, characterized by their large molecular weights, low copy numbers, and scarce unique restriction enzyme cutting sites. Through modifications such as the addition and optimization of components, natural plasmids can be transformed into the most commonly used vectors in genetic engineering, also known as plasmid vectors.

Functional Characteristics

Basic Components:

(1) Origin of Replication (Ori): A specific sequence rich in ATs and repetitive sequences that initiates plasmid replication by recruiting replication-related proteins. The Ori determines the copy number of the plasmid in the host; high-copy plasmids (10-60 copies) are known as relaxed-replication plasmids, whereas low-copy plasmids (1-3 copies) are designated as stringent-replication plasmids, often suited for cloning toxic genes and large gene fragments. A single Ori indicates a prokaryotic cloning or expression plasmid; two Ori's suggest a shuttle plasmid capable of replicating in both prokaryotic and eukaryotic systems. Note: Plasmids with identical Ori's are incompatible and cannot be co-transfected.

(2) Resistance Selection Gene (R): Also known as an antibiotic resistance gene, which facilitates subsequent selection of positive clones through antibiotic screening. Typically, cloning vectors have a single resistance selection marker, while some shuttle plasmids possess two. Note: Selection genes differ between prokaryotes and eukaryotes. Common in prokaryotes are Ampr, Camr, Kanr, Tetr, etc.; while Puro, G418, Hygr are prevalent in eukaryotes. Zeocin and Blasticidin can be used in both prokaryotes and eukaryotes.

(3) Multiple Cloning Site (MCS): A region containing multiple restriction enzyme cleavage sites, and each site is unique within the entire plasmid vector. The MCS serves as the insertion site for foreign genes, typically located between the promoter and transcription termination signals. Different vectors have varying types and numbers of restriction enzymes in their MCS. Note: Inserting excessively long gene sequences can reduce plasmid vector transformation efficiency.

(4) Promoter (P): A DNA sequence that initiates downstream DNA transcription by specifically binding to RNApol, without being transcribed itself. The promoter determines the cell type and expression level of the gene. Based on their expression patterns, promoters are classified into three types: constitutive/ubiquitous, tissue/cell-specific, and inducible promoters.

(5) Enhancer: A cis-acting element that enhances the transcription of adjacent genes, with no directionality.

(6) Ribosome Binding Site (RBS): Located upstream of the start codon (AUG) in mRNA, where ribosomes recognize and bind to initiate protein translation by searching downstream for ATG. In prokaryotes, the RBS on expression vectors is the SD sequence preceding AUG. In eukaryotes, reliance is primarily on the 5’ cap structure of mRNA; additionally, IRES elements and 2A polypeptide elements can act as RBSs to initiate protein expression.

(7) Transcription Terminator: Located downstream of the gene to be transcribed, after the 3’ regulatory elements, primarily consisting of polyadenylation or poly(A) signals, which terminate transcription. Prokaryotes mainly use polyA; eukaryotes, such as mammals, commonly employ terminators (SV40 polyA, hGH polyA, BGH polyA, and rbGlob) containing the sequence motif AAUAAA that promotes both polyadenylation and termination.

(8) Primer Binding Site (PBS): A short single-stranded DNA sequence that binds to PCR amplification or sequencing primers, primarily used for manual detection of plasmid sequences.

Classification of plasmid vectors

Plasmid vectors facilitate the introduction of genes of interest into host cells, enabling the expression of their functional capabilities. These vectors play a pivotal role in molecular biology research and serve as the foundation for advancing fields such as synthetic biology, biomedicine, gene therapy, target drug screening, and agricultural biotechnology. Given the diverse applications of plasmid vectors, their classification is equally varied, with the following primary categories based on their attributes:

1.

Viral Vectors vs. Non-Viral Vectors: According to their nature, plasmid vectors can be classified into viral and non-viral vectors. Viral vectors utilize viruses as gene delivery agents, typically by inserting or replacing the viral genome with a foreign gene to transport the desired gene into target cells. Viral vectors exhibit high infectivity and specificity, enabling efficient delivery and expression of exogenous genes within host cells.

Applications of Viral Vectors:

(1) Gene Therapy: Viral vectors are utilized to introduce therapeutic genes into target cells for the treatment of genetic disorders, cancers, and other diseases. This approach enables the delivery of corrective genetic material directly to affected cells.

(2) Gene Editing: Viral vectors serve as vehicles to transport gene-editing tools into target cells. This facilitates precise editing and modification of the genome, enabling scientists to correct genetic errors or introduce desired genetic changes.

(3) Vaccine Development: Viral vectors are employed as platforms for vaccine delivery, wherein they carry the gene encoding the target antigen into immune cells. This triggers an immune response, leading to the production of protective immune responses that can confer immunity against the targeted disease.

(4) Genetic Research: In the fields of gene function research, protein expression, and drug screening, viral vectors are used to transfer specific genes into cells or animal models. This approach enables the investigation of gene functions and mechanisms of action, facilitating a deeper understanding of biological processes and facilitating the discovery of new therapeutic targets and drugs.

The classification and comparison of viral vectors are as follows :

2.

Depending on the type of recipient cells they enter, vectors can be classified into prokaryotic vectors, eukaryotic vectors, and shuttle vectors (which can exist in both prokaryotic and eukaryotic cells).

3.

Based on the Ori element, vectors can be categorized into high-copy-number plasmids and low-copy-number plasmids.

4.

According to their nature, vectors can be divided into fusion vectors and non-fusion vectors. Fusion vectors are capable of integrating foreign genes into the genome of host cells, enabling stable expression of the foreign genes in the host cells. They are also known as stable expression vectors. In contrast, non-fusion vectors carry the target gene in a free-floating form within the cell, leading to transient expression of the target gene in the host cells. Hence, they are also referred to as transient expression vectors.

The comparison between transient expression vector and stable expression vector is as follows :

5.

Based on their different functions, vectors can be divided into cloning vectors, expression vectors, gene editing vectors, expression regulation vectors, in vitro transcription vectors and function detection vectors.