Shop All Gene Synthesis & DNA Assembly Kits

GeneArt™ Seamless PLUS Cloning and Assembly Kit (Invitrogen™)

Like our first generation Seamless Cloning and Assembly Kit, GeneArt® Seamless PLUS Cloning and Assembly Kit is the complete kit for simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute or less, room temperature reaction. However, Seamless PLUS offers several advantages over previous kits:

Increased Efficiency: pre-cloning option for large fragments for increased cloning efficiency
Larger Constructs: create constructs up to 40 kb
Versatility: high capacity, broad-range conjugative vector that replicates in most Gram negative bacteria

The improvements above are combined with these key benefits shared by all GeneArt® Seamless Cloning and Assembly kits:

Speed and Ease—clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector; no restriction digestion, ligation, or recombination sites required
Precision and Efficiency—designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Free Tools—design your final construct and DNA oligos in silico using our free web-based tool that takes you step-by-step through your project
• Vector Flexibility—use our linear vector or a vector of your choice
Diverse Applications—streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning of more than 4 DNA fragments or for final molecules larger than 110 Kb, please consider the GeneArt® High-Order Genetic Assembly System.

Simple and Fast Clone Creation
GeneArt® Seamless PLUS Cloning is a simple, two-step process, consisting of in vitro assembly followed by transformation into One Shot® DH10B™ T1R SA competent E. coli. The kit employs a proprietary enzyme/buffer mix to assemble DNA fragments with shared terminal end homology without extra sequences or scars in the final construct ("seamless"). Terminal end homology is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Seamless PLUS Cloning and Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA fragments (100 bp to 10 Kb), the total size of the final molecule (≤ 40 Kb), and the quality and specificity of each fragment.

Typical cloning efficiencies for different numbers of fragments:

• >95% for 4 fragments, 5 Kb each
• >90% for 4 fragments, 10 Kb each

Cloning success is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or PCR. The circularized clones obtained from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Design Support
A key step in GeneArt® Seamless PLUS Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. We provide a free online tool, the GeneArt® Design Tool for Seamless or High-Order Assembly and Mutagenesis, to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos with end homology for the PCR amplification of the different elements to clone, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® Seamless PLUS Cloning and Assembly Kit is designed to empower cloning and DNA assembly in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to construct fusion proteins; delete, replace, or add DNA elements such as restriction sites in an existing vector; and carry out many other techniques that require manipulation of genetic sequences.

GeneArt™ Gene Synthesis Kit (Invitrogen™)

The new GeneArt® Gene Synthesis Kit with CorrectASE™ technology provides you with all of the proven, high quality reagents necessary for successful do-it-yourself gene synthesis. CorrectASE™ enzyme removes mismatches caused by oligonuceotides errors and facilitates a 3–10 fold reduction of mutations in your synthetic genes or fragments.

The GeneArt® Gene Synthesis Kit:

• Increases the success rate of do-it-yourself gene synthesis by providing standardized reagents and protocol for the complete gene synthesis workflow
• Increases by 3–10 fold the probability of isolating a synthetic gene with correct sequence by including an error correction step into the workflow
• Enables gene synthesis in 3 days, from oligo assembly to sequence verified clone
• Reduces your labor time and sequencing costs by sequencing only 2–4 instead of 10–16 clones

Complete Workflow Solution for Do-it-yourself Gene Synthesis
The GeneArt® Gene Synthesis Kit contains all reagents required for successful do-it-yourself gene synthesis, including:

• High-fidelity Platinum® Pfx DNA Polymerase with automatic hot start for oligonucleotide assembly and amplification
• Quant-iT™ PicoGreen® dsDNA Reagent , an ultra-sensitive, fluorescent nucleic acid stain for quantification of double-stranded DNA (dsDNA)
• pCR™-Blunt II- TOPO® vector for synthetic gene cloning
• CorrectASE™ enzyme, our sequence error correction technology

Prevent Unwanted Mutations
Commercially available synthetic oligonuceotides have a high error rate during synthesis, ranging from one every 300–1000 bases, depending on the source. These errors cause frameshift (deletion and insertion) and mismatch mutations during gene synthesis. Incubation with CorrectASE™ enzyme removes both types of mutations.

The incubation step with CorrectASE™ enzyme is introduced after the initial PCR assembly of oligonucleotides. The PCR product is denatured and reannealed so that any mutations will be unmatched. CorrectASE™ enzyme binds to the resulting mismatches and nicks both DNA strands 3’ of the error. The 3’to 5’ exonuclease activity of the enzyme removes the errors. A final PCR with a proofreading polymerase then assembles the corrected fragments, thus increasing the likelihood of isolating clones with the correct sequence. Depending upon the incoming oligonuceotide quality, only 2–4 clones need to be screened, compared to 10–16 clones in a workflow that does not include the correction step. Including CorrectASE™ enzyme in your gene synthesis workflow decreases labor time and sequencing costs.

For Research Use Only. Not for animal or human therapeutic or diagnostic use.

GeneArt™ Type IIs Assembly Kit, Aar I (Invitrogen™)

The GeneArt® Type IIs Assembly Kit, Aar I, provides seamless cloning and assembly of up to 8 DNA fragments by simultaneous cleavage and ligation in a single reaction. The kit uses a technology similar to Golden Gate cloning with the type IIs restriction enzyme Aar I and can be used to assemble multiple fragments in a pre-determined order into any compatible vector. Since type IIs assembly is not based upon homologous recombination, there is minimal risk of rearrangements and minimal sequence confirmation of your final construct is required.

The kit is offered in three versions, each with a different type IIs restriction enzyme (Aar I, Bsa I, or Bbs I). All versions include all-in-one enzyme mix, cloning vector, and cloning controls. Our GeneArt® Primer and Construct Design Tool should be used to determine the appropriate GeneArt® assembly kit for your fragments; it provides an easy-to-use interface to design your construct and create and order primers, should they be required.

• Assemble multiple DNA fragments in any order, into any compatible vector, without scars
• Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
• Use for assembly of TALs, gene variants, and repetitive sequences
• Create your own cloning and expression vectors with custom vector elements
• Minimize sequence confirmation of final construct
• Pick from three type IIs enzymes

Type IIs Cloning
GeneArt® Type IIs Cloning is a simple, two-step process, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. The type IIS restriction endonuclease Aar I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Aar I recognition site. The ends of your DNA fragment can be designed to be flanked by the Aar I restriction site such that digestion of the fragments removes the recognition site and generates complementary overhangs that can be ligated seamlessly, creating a junction that lacks the original site. The kit includes a destination vector, called pType IIs, which is optimized for this approach.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Type IIs Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA elements, the total size of the final molecule (? 10 kb), and the quality and specificity of the fragment.

Typical cloning efficiencies for different numbers of fragments cloned into pType IIs are:

• >95% for 5 fragments of 1 kb each
• >60% for 8 fragments of 1 kb each
• >85% for 2 identical fragments of 1 kb each

in silico Cloning Design Support
A key step in GeneArt® Type IIs Cloning is the correct design of fragments and oligos with the appropriate recognition sites and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide the GeneArt® Primer and Construct Design Tool to help you design your experiment in silico. The tool recommends the correct GeneArt® kit for your assembly, checks for compatibility of the experimental design with the product specifications, designs DNA oligos, if needed, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with VectorNTI® software.

Additional recommended products:

• Zero Blunt® TOPO® PCR Cloning Kit, without competent cells (Cat. No. 450245)
• Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Cat. No. K280020)
• One Shot® MAX Efficiency™ DH10B™ T1 Phage Resistant Cells (Cat. No. 12331-013)

GeneArt™ Type IIs Assembly Kit, Bbs I (Invitrogen™)

The GeneArt® Type IIs Assembly Kit, Bbs I, provides seamless cloning and assembly of up to 8 DNA fragments by simultaneous cleavage and ligation in a single reaction. The kit uses a technology similar to Golden Gate cloning with the type IIs restriction enzyme Bbs I and can be used to assemble multiple fragments in a pre-determined order into any compatible vector. Since type IIs assembly is not based upon homologous recombination, there is minimal risk of rearrangements and minimal sequence confirmation of your final construct is required.

The kit is offered in three versions, each with a different type IIs restriction enzyme (Aar I, Bsa I, or Bbs I). All versions include all-in-one enzyme mix, cloning vector, and cloning controls. Our GeneArt® Primer and Construct Design Tool should be used to determine the appropriate GeneArt® assembly kit for your fragments; it provides an easy-to-use interface to design your construct and create and order primers, should they be required.

• Assemble multiple DNA fragments in any order, into any compatible vector, without scars
• Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
• Use for assembly of TALs, gene variants, and repetitive sequences
• Create your own cloning and expression vectors with custom vector elements
• Minimize sequence confirmation of final construct
• Pick from three type IIs enzymes

Type IIs Cloning
GeneArt® Type IIs Cloning is a simple, two-step process, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. The type IIS restriction endonuclease Bbs I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Bbs I recognition site. The ends of your DNA fragment can be designed to be flanked by the Bbs I restriction site such that digestion of the fragments removes the recognition site and generates complementary overhangs that can be ligated seamlessly, creating a junction that lacks the original site. The kit includes a destination vector, called pType IIs, which is optimized for this approach.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Type IIs Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA elements, the total size of the final molecule (? 10 kb), and the quality and specificity of the fragment.

Typical cloning efficiencies for different numbers of fragments cloned into pType IIs are:

• >95% for 5 fragments of 1 kb each
• >60% for 8 fragments of 1 kb each
• >85% for 2 identical fragments of 1 kb each

in silico Cloning Design Support
A key step in GeneArt® Type IIs Cloning is the correct design of fragments and oligos with the appropriate recognition sites and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide the GeneArt® Primer and Construct Design Tool to help you design your experiment in silico. The tool recommends the correct GeneArt® kit for your assembly, checks for compatibility of the experimental design with the product specifications, designs DNA oligos, if needed, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with VectorNTI® software.

Additional recommended products:

• Zero Blunt® TOPO® PCR Cloning Kit, without competent cells (Cat. No. 450245)
• Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Cat. No. K280020)
• One Shot® MAX Efficiency™ DH10B™ T1 Phage Resistant Cells (Cat. No. 12331-013)

GeneArt™ Type IIs Assembly Kit, Bsa I (Invitrogen™)

The GeneArt® Type IIs Assembly Kit, Bsa I, provides seamless cloning and assembly of up to 8 DNA fragments by simultaneous cleavage and ligation in a single reaction. The kit uses a technology similar to Golden Gate cloning with the type IIs restriction enzyme Bsa I and can be used to assemble multiple fragments in a pre-determined order into any compatible vector. Since type IIs assembly is not based upon homologous recombination, there is minimal risk of rearrangements and minimal sequence confirmation of your final construct is required.

The kit is offered in three versions, each with a different type IIs restriction enzyme (Aar I, Bsa I, or Bbs I). All versions include all-in-one enzyme mix, cloning vector, and cloning controls. Our GeneArt® Primer and Construct Design Tool should be used to determine the appropriate GeneArt® assembly kit for your fragments; it provides an easy-to-use interface to design your construct and create and order primers, should they be required.

• Assemble multiple DNA fragments in any order, into any compatible vector, without scars
• Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
• Use for assembly of TALs, gene variants, and repetitive sequences
• Create your own cloning and expression vectors with custom vector elements
• Minimize sequence confirmation of final construct
• Pick from three type IIs enzymes

Type IIs Cloning
GeneArt® Type IIs Cloning is a simple, two-step process, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. The type IIS restriction endonuclease Bsa I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Bsa I recognition site. The ends of your DNA fragment can be designed to be flanked by the Bsa I restriction site such that digestion of the fragments removes the recognition site and generates complementary overhangs that can be ligated seamlessly, creating a junction that lacks the original site. The kit includes a destination vector, called pType IIs, which is optimized for this approach.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Type IIs Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA elements, the total size of the final molecule (up to 13 kb), and the quality and specificity of the fragment.

Typical cloning efficiencies for different numbers of fragments cloned into pType IIs are:

• >95% for 5 fragments of 1 kb each
• >60% for 8 fragments of 1 kb each
• >85% for 2 identical fragments of 1 kb each

In Silico Cloning Design Support
A key step in GeneArt® Type IIs Cloning is the correct design of fragments and oligos with the appropriate recognition sites and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide the GeneArt® Primer and Construct Design Tool to help you design your experiment in silico. The tool recommends the correct GeneArt® kit for your assembly, checks for compatibility of the experimental design with the product specifications, designs DNA oligos, if needed, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with VectorNTI® software.

Additional recommended products:
• Zero Blunt® TOPO® PCR Cloning Kit, without competent cells (Cat. No. 450245)
• Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Cat. No. K280020)
• One Shot® MAX Efficiency™ DH10B™ T1 Phage Resistant Cells (Cat. No. 12331-013)

GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Invitrogen™)

GeneArt® Seamless Cloning and Assembly Enzyme Mix enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. GeneArt® Seamless enzyme mix is the economical choice for creating constructs up to 13 kb with the option for high-throughput assembly.

Efficiency: pre-cloning option for large fragments for increased cloning efficiency
Speed and Ease: clone DNA fragments, with sequence of your choice, in a single vector (up to 13 kb); no restriction digestion, ligation, or recombination sites required
Free Tools: design of your final construct and DNA oligos in silico using our free web-based tool that takes you step-by-step through your project
Vector Flexibility: use our linear vector or a vector of your choice

For the cloning of pre-existing DNA elements too long to be amplified by PCR and for final molecules up to 40 Kb, please consider the GeneArt® Seamless PLUS Cloning and Assembly Kit (1–4 DNA fragments only); and for final molecules larger than 110 Kb, please consider the GeneArt® High-Order Genetic Assembly System.

Simple and Fast Clone Creation
GeneArt® Seamless Cloning is a simple, two step process, consisting of in vitro assembly followed by transformation. The enzyme mix uses a proprietary enzyme/buffer mix to assemble DNA fragments without extra sequences or scars ("seamless"). Terminal end homology, if needed, is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

Cloning Efficiency, Flexibility, and Precision
Cloning success is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or PCR. The circularized clones obtained from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Cloning Design Support
A key step in GeneArt® Seamless Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. To simplify and speed the design process, we provide a free online tool, the GeneArt® Design Tool for Seamless or High-Order Assembly and Mutagenesis, to help you design your experiment in silico. The tool guides the user through construct design, including fragment import, order, and orientation; checks for areas of homology and potential design issues; creates primers for pre-cloning or incorporating end homology between fragments, if needed; and generates final construct map files and sequence in Genbank format for download or import into Vector NTI® software.

Applications
The GeneArt® Seamless Cloning and Assembly Enzyme Mix is designed to empower cloning and DNA assembly in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the mix to construct fusion proteins; delete, replace, or add DNA elements such as restriction sites in an existing vector; and carry out many other techniques that require manipulation of genetic sequences.

For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.

GeneArt™ High-Order Genetic Assembly Systems, with yeast growth media (Invitrogen™)

The GeneArt® High-Order Genetic Assembly System is a highly efficient kit for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 Kbp in length, into any vector. The system relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This greatly reduces the in vitro handling of DNA and eliminates the need for enzymatic treatments, such as restriction and ligation, while allowing for precise fusions of DNA sequences. The kit contains materials for the transformation and purification from yeast, including yeast selective media, and competent E. coli for plasmid amplification of correct clones.

Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt® Seamless Cloning and Assembly Kit (cat # A13288).

Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt® High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.

Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit.

Considerations for Choosing a Cloning Vector
The GeneArt® High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt® pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt® High-Order Vector Conversion Cassette (cat#A13291).

Cloning Efficiency
In GeneArt® High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ are the following:
• >90% for 5 DNA fragments of 10 Kb each
• >90% for 10 DNA fragments of 5 Kb each
• >50% for 10 DNA fragments of 10 Kb each

Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
• >90% for 1 fragment of 10 Kb
• >75% for 2 DNA fragments of 10 Kb each

Design Your Cloning in silico
A key step in GeneArt® High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.

GeneArt™ High-Order Genetic Assembly System (Invitrogen™)

The GeneArt® High-Order Genetic Assembly System is a highly efficient kit for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 Kbp in length, into any vector. The system relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This greatly reduces the in vitro handling of DNA and eliminates the need for enzymatic treatments, such as restriction and ligation, while allowing for precise fusions of DNA sequences. The kit contains materials for the transformation and purification from yeast (no growth media), and competent E. coli for plasmid amplification of correct clones.

Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt® Seamless Cloning and Assembly Kit (cat # A13288).

Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt® High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.

Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction step yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit. For selection of recombinant yeast clones we offer a selective yeast media kit, CSM Media for Mav203 Yeast Cells (cat# A13292).

Considerations for Choosing a Cloning Vector
The GeneArt® High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt® pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt® High-Order Vector Conversion Cassette (cat#A13291).

Cloning Efficiency
In GeneArt® High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ are the following:
• >90% for 5 DNA fragments of 10 Kb each
• >90% for 10 DNA fragments of 5 Kb each
• >50% for 10 DNA fragments of 10 Kb each

Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
• >90% for 1 fragment of 10 Kb
• >75% for 2 DNA fragments of 10 Kb each

Design Your Cloning in silico
A key step in GeneArt® High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.

GeneArt™ Seamless Cloning and Assembly Kit (Invitrogen™)

The GeneArt® Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.

Speed and Ease — Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); no restriction digestion, ligation or recombination sites required
Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Vector Flexibility — Use our linear vector or a vector of your choice
Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning more than 4 DNA fragments, final molecules larger than 110 Kb, or for using pre-existing DNA elements too long to be amplified by PCR; please consider the GeneArt® High-Order Genetic Assembly System (cat# A13285).

Simple and Fast Clone Creation
GeneArt® Seamless Cloning is a simple, two step process, consisting of a tube based assembly reaction followed by transformation into One Shot® Chemically Competent TOP10 E. coli. The kit uses the properties of a proprietary enzymatic mix to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). A portion of the assembly reaction is then transformed into the provided competent TOP10 cells, yielding clones ready for analysis the next day. The required 15-base pair end-homology can be easily engineered by PCR-amplification with custom DNA oligos.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Seamless Cloning and Assembly Kit the main factors effecting cloning efficiency are the size of the DNA elements (100 bp to 5 Kb), the total size of the final molecule (≤ 13 Kb), and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments cloned into pUC19L are the following:
• 90% for a single 5 Kb DNA element
• 70% for 4 fragments of 1 Kb each
• 40% for 4 DNA fragments of 2 Kb each

Success of the cloning is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or by PCR. The circularized clones obtain from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Cloning Design Support
A key step in GeneArt® Seamless Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide a free online tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos with end-homology for the PCR amplification of the different elements to clone, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® Seamless Cloning and Assembly Kit is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites in an existing vector, and many other techniques that require manipulation of genetic sequences.