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 For more information visit: https://products.invitrogen.com/ivgn/product/A13288?CID=cloningkit-SS-12312

1. GeneArt® Seamless Cloning and Assembly System
The GeneArt® Seamless Cloning and Assembly System enables cloning of up to 4 DNA fragments simultaneously into virtually any linearized vector,
typically in 30 minutes, without extra DNA sequences, restriction endonucleases, or ligation.
Product Quantity Cat. No.
GeneArt Seamless Cloning and Assembly Kit
®
20 reactions A13288
Component Size Quantity Component Size Quantity
10X Enzyme Mix 45 μL/tube 1 tube One Shot TOP10 Chemically Competent E. coli
®
50 μL/tube 21 tubes
5X Enzyme Buffer 90 μL/tube 1 tube pUC19 Control (10 pg/μL) 10 μL/tube 1 tube
Linear pUC19L Vector (50 ng/μL), 4 control reactions 8 μL/tube 1 tube S.O.C Medium 6 mL/bottle 1 bottle
Control insert (50 ng/μL) 5 μL/tube 1 tube
GeneArt® Linear pUC19L Vector for Seamless Cloning 20 reactions A13289
GeneArt® High-Order Genetic Assembly System
The GeneArt® High-Order Genetic Assembly System is a highly efficient, vector-independent system for the simultaneous and seamless assembly of
up to 10 DNA fragments (preexisting or synthetic) in vivo, totaling up to 110 kb in construct size.
Product Quantity Cat. No.
GeneArt® High-Order Genetic Assembly System 10 reactions A13285
GeneArt® High-Order Genetic Assembly System (with Yeast Growth Medium) 10 reactions/2 L medium A13286
GeneArt® High-Order Linear pYES1L Vector 10 reactions A13287
CSM Medium for Mav203 Yeast Cells 2L A13292
GeneArt High-Order Vector Conversion Cassette
®
10 reactions A13291
GeneArt® Site-Directed Mutagenesis System
The GeneArt® Site-Directed Mutagenesis System is a simple and highly efficient method for in vitro site-directed mutagenesis. The system can gener-
ate base substitutions, deletions, or insertions in DNA plasmids from any source, without specialized vectors, host strains, or restriction sites.
Product Quantity Cat. No.
GeneArt® Site-Directed Mutagenesis System 16 reactions A13282
Component Size Component Size Component Size
DNA Methylase (4 units/μL) 20 μL pUC19WHITE Control Plasmid (20 ng/μL) 100 ng 10X Enzyme Mix 45 μL
200X SAM (S-adenosine methionine) 10 μL Control Primer Mix (10 μM) 25 μL One Shot MAX Efficiency DH5α T1
® ® ™ R
1 box
10X Enhancer 100 μL PCR Water 1.8 mL
0.5 M EDTA 500 μL 5X Reaction Buffer 90 μL

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3. geNeTic AssembLy
headline essence
designedbrow you
headline to help
take control
GeneArt® Genetic Assembly Tools

4. GeneArt® Seamless Cloning and Assembly System
The geneArt® seamless cloning and Assembly system enables cloning of up to 4 DNA fragments simultaneously into
virtually any linearized vector, typically in 30 minutes, without extra DNA sequences, restriction endonucleases, or ligation.
• Speed—clone up to 4 DNA fragments (up to
13 kb total size) simultaneously in a single
tube, at room temperature, typically in 30
minutes
• Flexibility—use our linear vector or any GeneArt® Seamless Cloning and Assembly System workflow
vector of your choice
• Precision and efficiency—no extra Design PCR oligos using
DNA Oligo Designer, and
sequences; just clone what you want where PCR-amplify the DNA fragments
you want it Linear vector to add the required homology
DNA fragments
• Simplicity—online DNA oligo Designer
designs oligos and assembles DNA
molecules in silico
The GeneArt® Seamless Cloning and
Assembly System uses a proprietary enzyme
mix to recognize and precisely assemble
DNA fragments sharing a 15–base pair X
X
X Incubate 30 minutes at
end homology. End homology is created by room temperature
PCR amplification using primers designed DNA fragments and
linear vector recombine
to generate overlap between adjacent DNA
6–8 L of the
fragments to be assembled. reaction mix
One Shot® TOP10
The online DNA Oligo Designer Chemically
Competent E. coli Incubate 20–30 minutes on ice
(www.invitrogen.com/designdnaassembly) Heat shock for 30 seconds
guides users through experimental design, Add 250 L SOC
Recover for 1 hour at 37°C
including oligo design and ordering.
1 kbp fragments, 15 bp overlaps, 10–50 µL of the
AccuPrime™ Pfx polymerase transformation mix
120
GeneArt® Seamless Cloning and Assembly System
Product ‘I’ Plate on selective LB plates
100 Incubate overnight at 37°C
Assembled
Cloning efficiency (%)
80 circular construct
60
Pick 4–10 colonies
40
Isolate the assembled construct
20 from 4–10 positive colonies
Analyze by restriction digest
0
and/or sequencing
1 2 3 4
Number of inserts
Figure 1. The GeneArt® kit demonstrates higher
cloning efficiency than product “I” when more than
two inserts are cloned simultaneously into a linear
vector. The two kits were compared for cloning
efficiency using the same vector and inserts in four
cloning reactions. 1: one insert and vector; 2: two
inserts and vector; 3: three inserts and vector; 4:
four inserts and vector.

5. GeneArt® High-Order Genetic Assembly System
The geneArt® High-order genetic Assembly system is a highly efficient, vector-independent system for the simultaneous
and seamless assembly of up to 10 DNA fragments (preexisting or synthetic) in vivo, totaling up to 110 kb in construct size.
• Speed—clone 10 or more DNA fragments 100
simultaneously into a single vector (up to 110 kb
80
total); assemble existing DNA fragments without
Cloning efficiency (%)
restriction digestion or pcR amplification 60
• Flexibility—use our linear vector or a vector of your 40
choice; oligonucleotide stitching feature allows
end-editing and reuse of preexisting DNA fragments 20
without the need for reamplification 0
1 3 10
• Precision and efficiency—no extra sequences; just Number of 10 kb DNA fragments
clone what you want where you want it Figure 3. Cloning efficiency of the GeneArt® High-Order
Genetic Assembly System with increasing numbers of 10
• Simplicity—online DNA oligo Designer walks you kb PCR fragments. Even as 100 kb of PCR fragments are
through your project step by step; design your DNA assembled, the cloning efficiency remains over 65%, proving
the system is a reliable solution for assembly of complex DNA
oligos and assemble your DNA molecule in silico
molecules.
(www.invitrogen.com/designdnaassembly)
The GeneArt® High-Order Genetic
Assembly System relies on yeast’s
ability to take up and recombine A Fragment Oligo Cloning
DNA fragments with high efficiency size size efficiency
via transformation-associated
recombination, greatly reducing in x x x x
1 x 1 kb 60-mer 94%
x x x x
vitro handling of DNA and eliminating
the need for restriction digestion and x x x x x x
2 x 5 kb 80-mer 75%
ligation. x x x x x x
In cases where DNA fragments do x x x x x x x x [3 x 5 kb] + [2 x 0.5 kb] 60-mer 37%
not share end homology (i.e., existing x x x x
[3 x 5 kb] + [2 x 0.5 kb] 80-mer 75%
DNA fragments not created by PCR),
they can be “stitched” together using
B
recombinant linkers that provide 80-mer
2 x 5 kb 63%
sequence overlaps to promote
v
x x x x x x 10 bp insertion
recombinatorial joining of unrelated x x x
v
x x x
2 x 5 kb 80-mer 50%
DNA fragments (Figure 2A). 20 bp insertion
x x x x x x 80-mer
In addition, oligonucleotide stitch- x x x x x x
2 x 5 kb
12 bp insertion
87%
ing allows editing of the fragment
junctions to generate deletions and Figure 2. Oligonucleotide stitching. (A) Oligonucleotide stitching enables joining
insertions (Figure 2B). This feature of unrelated DNA fragments. (B) Oligonucleotide stitching can be used for junction
also allows the reuse of existing DNA editing.
fragments or elements obtained by
restriction enzyme digestion without
the need to reamplify them by PCR.

6. GeneArt® Site-Directed Mutagenesis System
The geneArt® site-Directed mutagenesis system is a simple and highly efficient method for in vitro site-directed
mutagenesis. The system can generate base substitutions, deletions, or insertions in DNA plasmids from any source, without
specialized vectors, host strains, or restriction sites.
• Control—insert, delete, or change up to 12
nucleotides in plasmids up to 14.5 kb
GeneArt® Site-Directed Mutagenesis System protocol
• Speed—entire protocol, including
transformation, is completed typically in less
than 3 hours (using a 3 kb plasmid) Plasmid template
PCR Reagents
Methyl Transferase Reagents
• Precision and efficiency—mutagenesis
efficiency over 90% (using a 3 kb plasmid)
Methylation
The GeneArt® Site-Directed Mutagenesis
System utilizes mutagenic oligonucleotide
Methylated
plasmid Step 1:
Methylation
primers to generate mutations. The mutagen-
esis protocol is streamlined by combining DNA x
x Mutagenesis
methylation and amplification steps into a single x
reaction, eliminating post-mutagenesis diges- PCR sample
x
tion and purification steps. 10X enzyme mix
Reaction buffer
The GeneArt® Site-Directed Mutagenesis
System delivers superior mutagenesis per- Step 2:
formance with a wide range of vector sizes. A Recombination reaction
x
x
comparative analysis against competitor “Q” x
x
reveals the advantage of using the GeneArt®
In vitro Add 1 L 0.5 M EDTA to stop the reaction
Site-Directed Mutagenesis System for a single– recombination Use 2 L of recombinant reaction sample
reaction for transformation
base pair mutation (Figure 4). x
x
x
x
Step 3:
As the size of the template plasmid increases, x
Transformation
the superior mutagenesis efficiency of the
GeneArt® system is clearly demonstrated.
Transformation Incubate on ice for 15 minutes
The flexibility, efficiency, and speed make the Heat-shock for 30 seconds
GeneArt® Site-Directed Mutagenesis System Add 250 L SOC medium
Recover for 1 hour at 37°C
the ideal choice for your routine and complex
Mutated
site-directed mutagenesis needs. plasmid
100
GeneArt® Site-Directed
Mutagenesis System
Steps 1–3:
Mutagenesis efficiency (%)
90
Competitor ‘Q’ 1. Methylate plasmid DNA and amplify the plasmid in a mutagenesis reaction
with two overlapping primers containing the target mutation.
80
2. Perform the in vitro recombination reaction, which enhances the colony
output 3x to 10x.
70
3. Transform the sample into DH5α™-T1® competent E. coli. The host cell circularizes
the linear mutated DNA, and McrBC endonuclease in the host cell digests the
60
methylated template DNA, leaving only unmethylated mutated product.
50
2.8 5.9 8.9
Plasmid size (kbp)
Figure 4. GeneArt® Site-Directed Mutagenesis System
vs. competitor “Q”.