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ORF Cloning
ORF cloning precisely captures the protein-coding region from the start codon ATG to the stop codon while excluding the 5' and 3' UTRs. The resulting construct can be used directly for protein expression in mammalian cells, insect cells, or Escherichia coli.
We support three template sources: cDNA, genomic DNA, and synthetic DNA.
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  • 5-15 calendar days

    Standard turnaround time

  • 100%

    ORF sequence accuracy

  • 200+

    In-stock vectors

  • 5μg

    Standard plasmid DNA delivery

首页 > Products > Plasmid Construction > ORF Cloning >
Introduction
Product Comparison
Template Sources
Service Advantages
Service Details
Optional Add-On Services
Service Workflow
Delivery Standards
Quality Control Standards
Tag Selection Guide
Applications
Frequently Asked Questions
ORF Cloning
Subcloning
PCR Cloning

ORF Cloning

Precisely Capture Coding Regions for Efficient Protein Expression

ORF (Open Reading Frame) cloning inserts the protein-coding region of a gene, from the ATG start codon to the stop codon, into an expression vector. Unlike conventional cDNA cloning, ORF cloning removes the 5' and 3' untranslated regions (UTRs), avoiding potential effects of complex UTR secondary structures on translation efficiency and enabling direct protein expression in mammalian cells, insect cells, or E. coli.


We provide professional ORF-cloning and expression-vector construction services, backed by a large inventory of ready-to-use cDNA templates. Target ORF sequences can also be amplified from cDNA, genomic DNA, or synthetic DNA. Each ORF clone includes the NCBI gene identifier, gene aliases, ORF sequence, amino-acid sequence, cloning-vector information, and tag information.

Subcloning

Precise Transfer of Existing ORFs Between Vectors

Suitable for transferring an existing target ORF from its current vector into a new expression, tag, or shuttle vector. Extensive restriction-ligation experience and advanced seamless-cloning technology support restriction-site compatibility, long-fragment transfer, and multi-fragment assembly.


  • No restriction-site limitations;
  • Supports parallel subcloning of the same fragment into multiple vectors;
  • Precisely addresses reading-frame matching, tag fusion, and promoter replacement;
  • Use a customer-provided vector or choose from more than 200 available vectors.

PCR Cloning

A Direct Route from Template to ORF

Suitable for directly amplifying a target ORF from cDNA, genomic DNA, or an existing plasmid. High-fidelity polymerases and optimized reaction systems provide high sequence fidelity and specificity.


  • Supports templates including plasmid DNA, genomic DNA, cDNA, and PCR products;
  • Products can be cloned directly into T vectors or blunt-end vectors without restriction-site compatibility concerns；
  • Suitable for rapid ORF acquisition and preliminary validation before protein-expression studies.

ORF Cloning vs. cDNA Cloning: How to Choose

Choose ORF cloning for direct protein expression and cDNA cloning for a complete transcript template
Comparison Dimension ORF Cloning (This Service) cDNA Cloning
Sequence Scope Open reading frame only, from ATG to the stop codon, with UTRs removed Full-length transcript including 5'/3' UTRs
Vector Type Expression vector such as the pCMV series, with a strong promoter Cloning vector such as pUC19/pMD19-T
Purpose Direct cell transfection for protein expression PCR template, sequencing template, and gene-function studies
Tag Optional His, FLAG, HA, Myc, GFP, and other tags Usually untagged
Expression Verification Yes, verified by protein expression No, sequence verification only
Ready-to-Use Status Ready to use without subcloning Requires further subcloning into an expression vector
Kozak Sequence Included to improve translation efficiency Usually absent
Applications Protein overexpression, purification, localization, and stable cell-line generation Gene-cloning templates, qPCR, and library construction

Selection Guidance:Choose ORF cloning to express a target protein directly in cells for functional studies, purification, or localization. Choose cDNA cloning when only the gene sequence is needed as a PCR template or for basic research.

Template Sources

Choose flexibly based on template availability and experimental requirements
Template SourcesApplicationsFeatures
Ready-to-Use cDNA TemplatePopular genes available in the ready-to-use libraryFastest delivery, low cost, and verified sequence
Customer-Provided cDNACustomer cDNA library or plasmidSequencing verification is required and billed separately
Genomic DNAIntronless genes or prokaryotic genesDirect amplification from the genome without mRNA
Synthetic DNANo template available, or codon optimization/sequence modification requiredHigh customization flexibility with sequence design tailored to requirements

Service Advantages

One-stop service with ready-to-use templates, sequencing verification, stocked vectors, and flexible tag options
  • Ready-to-Use Templates · Rapid Start
    A ready-to-use cDNA library covering human, mouse, rat, rabbit, and other species, with support for cDNA, genomic DNA, or synthetic DNA as the source.
  • Sanger Sequencing Verification · Traceable Data
    The ORF region is verified by Sanger sequencing and matched to the confirmed reference sequence.
  • 200+ Ready-to-Use Vectors · Select and Start
    Supports fusion tags such as His, GST, FLAG, and GFP together with promoter combinations for direct cloning into expression vectors.
  • Expression Vectors · Ready-to-Use Design
    Uses expression vectors such as pCMV with an enhanced CMV promoter and Kozak consensus sequence, with the 5'/3' UTRs removed.
  • Multiple Tag Options · Flexible Applications
    Supports N- or C-terminal untagged, FLAG, HA, His, Myc, GFP, mCherry, and other tag options.

Service Details

Subcloning and PCR-cloning options cover vector transfer and template amplification

Subcloning Service:Transfer an ORF from an existing plasmid into the destination expression vector after restriction digestion or PCR amplification. Suitable for vector transfer, tag addition, promoter replacement, and related applications.

PCR Cloning Service:Use cDNA, genomic DNA, or synthetic DNA as the template, amplify the ORF by PCR, and clone it into an expression vector.

Gene Length Turnaround Time (Calendar Days) Price
< 3 kb 5 - 15
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3 kb ~ 4 kb 15 - 20
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4 kb ~ 5 kb 20 - 25
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> 5 kb Project Evaluation
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Optional Add-On Services

Tag addition, codon optimization, and plasmid preparation at multiple scales
Add-On ServiceDescriptionTurnaround
Tag AdditionAdd FLAG, HA, His, Myc, GFP, or other tags at the N or C terminus2–3 business days
Codon OptimizationOptimize codon usage for the expression host to improve protein yield1–2 business days
Endotoxin-Free Plasmid PreparationSuitable for transfection-grade plasmid requirements, with endotoxin <0.1 EU/μg3–5 business days
Large-Scale Plasmid PreparationMidi(>100μg) / Maxi(>500μg) / Mega(>2mg) / Giga(>10mg)5–7 business days

Service Workflow

Requirement submission, plan and quotation, experimental implementation, delivery, and support
  • Submit Requirements
    Provide the Gene ID, Accession Number, or gene sequence, and specify the vector, tag, restriction sites, and any special requirements.
  • Plan and Quotation
    Our technical team responds within one business day with feasibility, vector recommendations, the experimental plan, turnaround estimate, and quotation.
  • Experimental Implementation
    Complete ORF amplification, vector linearization, homologous recombination, transformation screening, and sequencing verification.
  • Delivery and Support
    Deliver the sequence-verified recombinant plasmid, glycerol stock, and complete report, with long-term technical support.

Delivery Standards

Complete delivery of plasmid, bacterial stock, sequence files, sequencing data, plasmid map, and quality report
  • Plasmid DNA
    Deliver 5 μg of high-purity plasmid DNA containing the target insert, supplied as lyophilized powder or liquid.
  • Glycerol Stock (Optional)
    Provide a glycerol stock containing the correct plasmid, stored at -80°C and ready for recovery when needed.
  • Standard Sequence Files
    Provide the final plasmid sequence in FASTA/GenBank format with the insert and key elements fully annotated.
  • Sequencing Results
    Provide raw Sanger-sequencing chromatograms (.ab1) and a complete sequence-alignment report.
  • Plasmid Map
    Provide a fully annotated plasmid map.
  • Certificate of Analysis (COA)
    Record key parameters including plasmid quantity, purity (A260/A280 ratio), and delivered amount.

Quality Control Standards

Multi-level quality control for every ORF-cloning batch under the ISO 9001 quality-management system
QC ItemTest MethodAcceptance Criteria
Sequence AccuracyFull-length bidirectional Sanger sequencingThe ORF region is verified by Sanger sequencing and matched to the confirmed reference sequence
Plasmid PurityUV spectrophotometryA260/A280 = 1.8 ~ 2.0
Plasmid QuantityUV absorbance≥5 μg (standard delivery amount)
Insert SizeColony PCR/restriction-digest verificationMatches the expected ORF insert size
Bacterial ViabilityGlycerol-stock recovery cultureNormal colony growth after recovery
Endotoxin LevelLAL assay (endotoxin-free grade)< 0.1 EU/μg

Tag Selection Guide

Different tags suit different applications; selecting the appropriate tag is critical to experimental success
Tag Position Applications Advantages
His (6xHis) N or C terminus Protein purification (IMAC) and Western blot detection Small tag with low risk of interfering with protein function;
simple and cost-effective purification
FLAG N or C terminus Protein purification and immunoprecipitation
(IP/Co-IP), and detection		 Small tag with high-affinity antibodies and low background
HA N or C terminus Immunodetection and immunoprecipitation Small tag with well-established, reliable antibodies
Myc N or C terminus Immunodetection and cellular localization Small and widely used detection tag
GFP / EGFP Usually C-terminal Live-cell localization, expression verification, and FRET Direct fluorescence observation without antibodies;
enables dynamic live-cell tracking
mCherry Usually C-terminal Live-cell localization and multicolor colocalization Red fluorescence for dual labeling with GFP
Untagged None When a tag may interfere with function or folding Closest to the native protein, but requires a suitable antibody for detection

Applications

Covering protein expression, functional studies, drug development, gene therapy, and synthetic biology

Recombinant Protein Expression and Purification

Clone the ORF into an expression vector containing a His, GST, FLAG, GFP, or other tag for efficient expression in HEK293, CHO, E. coli, or insect cells.

Gene Function and Signaling-Pathway Studies

Construct ORF-overexpression vectors to study cell proliferation, differentiation, apoptosis, and migration, and combine them with site-directed mutagenesis to analyze key residues.

Antibody and Drug-Target Development

Produce recombinant antigen proteins for monoclonal-antibody generation, immunodetection, vaccine development, and activity evaluation of candidate targets.

Gene and Cell Therapy

Clone therapeutic-gene ORFs into lentiviral, AAV, or other vectors for gene-replacement therapy or CAR-T product development.

Synthetic Biology and Protein Engineering

Used for metabolic-pathway reconstruction, enzyme engineering, and biosensor development, with support for modular assembly and high-throughput screening.

Frequently Asked Questions

Frequently asked questions about ORF selection, tags, expression verification, template sources, and vector conversion
What is ORF cloning, and how does it differ from cDNA cloning?
ORF (Open Reading Frame) cloning inserts the protein-coding region of a gene, from the ATG start codon to the stop codon, into an expression vector.
The key differences from cDNA cloning are:ORF cloning removes the 5' and 3' untranslated regions (UTRs) and retains only the coding sequence,It is cloned into an expression vector with a strong promoter, such as pCMV, and can be transfected directly into cells for protein expression.

cDNA cloning retains the complete transcript, including the UTRs, in a cloning vector such as pUC19. It is primarily used as a PCR template or for gene-function studies and requires further subcloning for protein expression.

In short, choose ORF cloning for protein expression and cDNA cloning for a gene template.
What are the benefits of removing the UTRs in ORF cloning?
It avoids inhibition of translation initiation by complex UTR secondary structures and makes the sequence more compact and easier to modify;
ORF clones usually include a Kozak consensus sequence (GCCACCAUGG) together with a strong promoter such as CMV.
Which tag should I choose, and should it be placed at the N or C terminus?
Tag selection depends on the experimental goal: choose His or FLAG for purification, GFP/mCherry for live-cell localization, and HA/Myc for immunodetection.

Principles for choosing the N or C terminus:
(1) If the protein has an N-terminal signal peptide or transmembrane region, place the tag at the C terminus to avoid interference;
(2) If the C terminus contains a functional domain, place the tag at the N terminus.
Can your ORF clones be transfected directly into cells?
Yes. ORFs are cloned into expression vectors such as pCMV with a strong CMV promoter and can be used directly for transient transfection of mammalian cells including HEK293, HeLa, and CHO.

For large-scale transfection or sensitive primary and stem cells, choose the endotoxin-free plasmid-preparation add-on. Endotoxin is controlled below 0.1 EU/μg for improved transfection performance.
What template sources are available for ORF cloning, and which should I choose?
Ready-to-use cDNA templates, genomic DNA, and synthetic DNA are supported. Choose a ready-to-use template when available;
If no ready-to-use template is available but a genomic sample is available, choose PCR cloning;
Choose synthetic DNA when codon optimization is required or no template is available.
Can ORFs larger than 5 kb be cloned?
Yes, subject to specialized evaluation of GC content, repeats, secondary structure, and other factors. The ORF may be obtained through gene synthesis or homologous recombination from a large-fragment vector such as BAC/PAC.
Can an ORF clone be transferred into a lentiviral vector?
Yes. The ORF can be transferred into a lentiviral expression vector such as pLV for transduction of difficult-to-transfect cells and generation of stable cell lines;
Lentiviral vector capacity is generally ≤8–10 kb, including the ORF and tag; very large ORFs require evaluation.

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