iDNA Next Generation Cloning

  1. + pRedET
  2. + pCre
  3. pSub-Hox-A11
  4. loxP
  5. (18 kb)
  6. loxP
  7. loxP
  8. PGK loxP neo loxP
  9. Hox-A11
  10. Conditional knockout
  11. targeting vector
  12. (19.5 kb)
  13. gb2
  14. pSub-Hox-A11
  15. loxPneo
  16. (19.5 kb) loxP
  17. PGK
  18. loxP
  19. neo
  20. gb2
  21. ORI amp
  22. Hox-A11
  23. homology region
  24. Hox-A11
  25. homology region
  26. minimal vector
  27. pSub-Hox-A11 (18 kb)
  28. + pRedET
  29. + pRedET
  30. Red/ET RecombinationCloning Without Restriction Enzymes
  31. A Guide to Next Generation Cloning
  32. pBAD
  33. Promoter
  34. Precise, High-Speed Cloning
  35. with Red/ET Recombination

iDNA Red/ET Recombination permits the engineering of DNA in
E. coli using homologous recombination mediated by phage
protein pairs, either RecE/RecT or Reda/Redb.

Recombineering with Red/ET differs from other DNA engineering technologies, such as cutting and pasting with
restriction enzymes, PCR, DNA ligase, because it is
• not limited by DNA size
• independent of restriction sites

Any DNA molecule in E. coli of almost any size can be engineered at any site using Red/ET.

Recombineering therefore permits more DNA engineering
freedom than any other technology and has become an essential component of the molecular biological tool kit.

Recombineering can easily be deployed alongside conventional DNA methodologies to give you time to do your research.

iDNA  applications of Red/ET

Why is Red/ET Recombination a superior approach to
DNA engineering?

Red/ET Recombination allows a faster, more flexible and highly
reliable modification of plasmids, BACs, or the E. coli genome than
conventional cloning methods.
Red/ET exploits phage l homologous recombination potential for
in vivo genetic engineering in E. coli. Since Red/ET does not depend on restriction enzymes, ligation reactions or in vitro cleanup steps, it is highly applicable for the engineering of large DNA

Animal targeting constructs

Red/ET allows for the genetic engineering of tailor-made targeting
constructs for animal models:

• conditional knock-out/knock-in
• promoter or reporter fusions
• exon swapping
• introduction of point mutations
strain modification
With Red/ET you can easily modify the E. coli genome:
• gene disruption, deletion or insertion
• reporter gene and tag integration
• promoter fine tuning
• introduction of point mutations
Red/ET Recombination
Red/ET at a glance
• only 50 bp of flanking sequence
sufficient for recombination
• sequence independent
• precise at any position
• cloning without restriction enzymes
• no ligation reactions
• cloning of inserts up to 80 kb
Selection Marker
PGK gb2

You can establish Red/ET Recombination

Red/ET Recombination

Methods And Compositions For Directed Cloning and Sub-cloning Using
Homologous Recombination.

Three Simple Steps with Red/ET
1. Attachment of homology arms
Red/ET Recombination requires linear DNA which is flanked by
terminal homology stretches of only 50 bp. Thus, DNA homology
arms for any given locus can easily be attached by PCR.
Heterologous DNA
50bp homology arms
2. Recombineering
The insert is introduced into E. coli cells propagating the Red/ET
expressing plasmid.
Red/ET mediated
3. Selection/screening
Cells harboring the recombinant DNA are selected.
Three simple steps
Cells which express l-derived red
genes from plasmid pRed/ET promote base precise exchange of DNA sequences flanked by homology arms.
The in vivo reaction is catalyzed by
the exonuclease Reda and the DNA
annealing protein Redb.

Even the most demanding tasks can
be reduced to three basic steps:

1. Attachment of Homology Arms
2. Recombineering
3. Selection/Screening
Zhang Y., Buchholz F., Muyrers J.P.P.
and Stewart A.F. A new logic for DNA
engineering using recombination in E.
coli. Nature Genetics 20 (1998) 123-

Muyrers, J.P.P., Zhang, Y., Testa, G.,
Stewart, A.F. Rapid modification of
bacterial artificial chromosomes by
ET-recombination. Nucleic Acids Res.
27 (1999) 1555-1557.
Zhang Y., Muyrers J.P.P., Testa G. and
Stewart A.F. DNA cloning by homologous recombination in E. coli. Nature
Biotechnology 18 (2000) 1314-1317.

Animal Targeting Constructs

How can Red/ET Recombination help you to make tailored constructs for animal models?

At the cutting edge of DNA engineering, Gene Bridges‘ kits and
cloning services fulfill the needs of our pharmaceutical, biotech,
and academic clients for tailor-made targeting constructs.

Transgenic technologies are an essential component in the study
of developmental biology and modelling genetic disorders.

Red/ET Recombination enables in vivo DNA modifications irrespective of composition and size. Thus, Gene Bridges´ recombination kits open up exciting possibilities for the fast and reliable
engineering of targeting constructs.

iDNA services:

• Assistance with project design and verification strategy
• Steps to improve efficiency for subsequent ES cell recombination or blastocyte microinjection
• Full documentation
Size of the constructs
Constructs can be prepared as high-copy plasmids with an overall
size of up to 30 kb or low-copy plasmids with a size of up to 50 kb.
Basic targeting construct
For efficient ES cell recombination, flanking homology arms can be extended to 5 kb (short arm) and 10 kb (long
arm). Appropriate restriction sites for linearization (L) and screening (S) can easily be incorporated.
Exon PGK gb2 Selection Marker
Long arm Short arm
ES cell recombination ES cell recombination
PGK gb2
Long Arm Short Arm

Animal Targeting Constructs
Conditional knock-out targeting constructs
Analyse gene function by flanking an essential exon with loxP
sites for subsequent excision by Cre recombinase.
Long arm Short arm
Exon PGK gb2 neo
Reporter constructs
Analyse gene expression by a reporter gene fused to the native
promotor without introducing additional nucleotides which may
affect the expression pattern.
P Reporter PGK gb2 neo Exon
Long arm Short arm
Promoter fusion constructs
Fuse your cDNA to the promoter of interest, without introducing
additional nucleotides which may affect the expression pattern.
P cDNA PGK gb2 neo Long arm Short arm
Custom service work flow for
targeting constructs
1. Provide us with the gene name (NCBI
Acc. No.) and kind of modification.
2. We develop an in silico strategy and
provide you with the electronic data
for cross checking.
3. We order the appropriate BAC clone
based on the mouse strains C57/BL6
or 129Sv.
4. We clone ≤18kb of the modified allele into a high-copy vector backbone, providing large (>5kb) homology arms for an efficient ES cell
recombination. Constructs ≥20kb
are available as low copy plasmids
upon request.
5. We confirm integrity of the final targeting construct by sequencing.
6. You receive an E. coli glycerol stock

Animal Targeting Constructs
Targeting constructs introducing point mutations
Study the effects of SNPs in your animal model and insert single
base pair mutations at any position.


Targeting constructs to humanize animal models
Replace a given exon with the human counterpart to study the
influence of your drug on a human-derived allele.
Murine Exon HumanExon Murine Exon
Long arm Short arm
Optimized transgene constructs
Transfer a whole genomic locus up to 50 kb from a BAC clone into
a low copy plasmid. Unwanted flanking sequences are removed
yielding optimized constructs.
Gene of interest
BAC clone Low-copy
Transgenes are generally more reliably expressed if the intronexon boundaries are preserved in the transgene construct.

How can Red/ET Recombination help you to optimize


Escherichia coli is frequently used as model organism and functions
as microbial factory in biotechnology. Recombination with Red/ET
enables a defined and rapid access to chromosomal modifications:
• Gene disruption, deletion, insertion, modification
• Reporter gene or tag integration
• Promoter fine tuning
Use the modular system of our recombination kits and functional
cassettes to prepare optimized E. coli strains or use our service
facility in Heidelberg, Germany.
Markerless knock-out of genes
In combination with Flp/Cre technology multiple markerless genome modifications can be achieved.
Strain Modifications
Custom service work flow for
strain optimization
1. Provide us with a reference sequence file and your E. coli strain*.
2. We will help to optimize the project
design and provide you with electronic data for cross checking.
3. We confirm clone integrity by sequencing.
4. You receive an E. coli glycerol stock

E. coli Genome

  1. FRT/loxP
  2. E. coli Genome
  3. FRT/loxP
  4. FRT/loxP FRT/loxP FRT/loxP
  5. Enter another modification
  6. cycle if appropriate
  7. Selection marker removal
  8. by Flp/Cre recombination
  9. FRT/loxP
  10. Screening for
  11. loss of marker

E. coli Genome

Seamless modifications
rpsL sm
Insert point mutations or other seamless modifications by employing a selection – counterselection cassette (rpsL-sm).
Reporter gene or tag integration
Gene X Reporter
Use a chromosomal reporter strain to analyse single copy gene
Promoter fine tuning
sm Gene X
Optimize gene expression by fusion of a synthetic promoter library (SPL) to the gene of interest.
Protein tagging in E. coli cells achieved by chromosomal fusion of GeneX with cfp, encoding
for cyan fluorescent protein. A. Fluorescence
microscopy. B. Dark field microscopy. Pictures
provided by Stavans Lab, Weizmann Institute of
Science, Israel.
Promoter Strength
Analysis of the activity of pgi-lacZ under control of a synthetic promoter library. A. Transformants appear white to dark blue in the presence of X-gal. B. Promoter activity of a subset
of SPL clone. Red: native promoter.
(Biotechniques, Vol 45, No 3, 2008).
Strain Modifications

How can you take advantage of large BAC libraries?
Genome projects for more than 400 eukaryotic organisms are currently either running or finished. For the majority of these projects,
annotated large insert BAC (bacterial artificial chromosome) libraries are available.
Red/ET Recombination makes this valuable source easily accessible.
Large genomic fragments of any sequence can be cloned into plasmids by gap repair. The method is not restrained by the general
fidelity and amplicon size limitations of PCR.

Advantages of BAC subcloning

• Fast and simple cloning of large fragments
• Cloning size up to 30 kb for high-copy plasmids
• Cloning size up to 80 kb for low-copy plasmids
• Cloning independent of restriction sites
• Flanking of the cloned fragment with functional cassettes or
restriction sites easily possible


ori sm
A plasmid backbone which contains an origin of replication (ori)
and a selectable marker (sm) is PCR-amplified with primers introducing homology arms (red).
Large Fragment Cloning
Selected BAC resource centres
BACPAC Resource Center at Children‘s
Hospital Oakland Research Institute
Arizona Genomics Institute
Clemson University Genomics Institute

MF 11
Red/ET Recombination Kits
Establish Red/ET Recombination in your lab – Gene Bridges offers
a wide range of kits, modular cassettes and plasmids.
Quick Easy BAC Modification Kit
Quick & Easy
BAC Modification Kit
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K001
Version 2.6 (Oc tober 2007)
Cat.No. K001
Developed for deletion of BAC fragments
and all types of basic modifications.
Functional elements: Tn5-neoR
Counter Selection BAC Modification Kit
BAC Modification Kit
(Advanced BAC Modification Kit)
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K002
Version 3.0 ( Januar y 2007)
Cat.No. K002
Developed for the insertion of point
mutations in BAC clones or the E. coli
genome. Functional elements: Positive/
negative selection marker cassette rpsLneoR
BAC Subcloning Kit
BAC Subcloning Kit
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K003
Version 2.5 (December 2006)
Cat.No. K003
Developed to transfer fragments up to
30 kb from BACs or the E. coli chromosome. Functional elements: Linear vector
ColE1 ori-ampR
Kit Contents
• pRed/ET expression plasmid
• Suitable control experiments
• Detailed manual, maps, sequences
Academic researchers can order Gene
Bridges kits, cassettes and plasmids

Red/ET Recombination Kits
Quick Easy Conditional Knock-Out Kit
Quick & Easy
Conditional Knockout Kit
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K004
Version 1.3 ( June 2007)
Quick & Easy
Conditional Knockout Kit
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K005
Version 1.1 ( June 2007)
Cat.No. K004 (loxP), K005 (FRT)
Developed for the insertion of
FRT or loxP sites, respectively into
high-copy plasmids. Flp or Cre expression plasmid included.
Functional elements:
loxP-PGK-gb2-neo-loxP (K004)
FRT-PGK-gb2-neo-FRT (K005)
Quick Easy Gene Deletion Kit
Quick & Easy
E. coli Gene Deletion Kit
By Red®/ET® Recombination
Technic al Protocol
Cat. No. K006
Version 2.1 (November 2007)
Cat.No. K006
Developed for the deletion of E.
coli genes. Markerless modification possible in combination with
expression plasmid A104 or A105
(see page 14).
Quick Easy RNAi Rescue Kit
Quick & Easy
RNAi Rescue Kit
By Red®/ET® Recombination
SNAP-tag™ TMR-Star Detection
Technic al Protocol
Cat. No. K007
Version 1.2 ( Januar y 2007)
Cat.No. K007
Developed to insert a SNAP-tag
cassette into a BAC clone to confirm the specificity of an RNAibased loss-of-function (LOF)
phenotype. Determine the transfection efficiency by the integrated SNAP-tag casette. amp
ORI amp
Hox-A11 homology region
(19.5 kb)
3. Red/ET Recombination
4. Cre – mediated Excision
5. Red/ET Recombination
2. Red/ET Recombination

(18 kb)
(18 kb) loxP
PGK neo loxP loxP
Conditional knockout
targeting vector
(19.5 kb)
+ loxP loxP
Hox-A11 homology region
minimal vector
loxP loxP
PGK – gb2 – neo

1. PCR
Constructing conditional knock-outs
Construction of a conditional knock-out targeting vector for the
murine homeobox protein Hox-A11 using the BAC Subcloning Kit
and the Quick Easy Conditional Knock-Out Kit (loxP/cre).

Increase your flexibility by using additional selection marker cassettes optimized for Red/ET recombination

Cassette Features
• Selection cassettes are driven by eukaryotic (PGK) and prokaryotic (gb2) promoters, respectively
• Selection markers flanked by loxP or FRT-sites can be removed
in a Cre or Flp recombination step where appropriate
• Due to a modular architecture, selection cassettes can be PCRamplified with master primers
• Zero background because cassettes are encoded by suicide plasmids to avoid false positive clones
Basic functional cassette

  1. loxP/FRT
  2. loxP/FRT
  3. PGK gb2
  4. T3 pA T7

Cassettes overview
Selection Cassettes

  1. PGK : Eukaryotic promoter
  2. gb2 : Prokaryotic promoter
  3. FRT : Flp recognition target site
  4. loxP : Cre recognition target site
  5. neo : Neomycin
  6. kan : Kanamycin
  7. cm : Chloramphenicol
  8. amp : Ampicillin
  9. hyg : Hygromycin
  10. A001 PGK-gb2-neo kanR neoR
  11. A002 FRT-PGK-gb2-neo-FRT kanR neoR
  12. A003 loxP-PGK-gb2-neo-loxP kanR neoR
  13. A004 FRT-PGK-gb2-neo-FRT-loxP kanR neoR
  14. A005 loxP-FRT-PGK-gb2-neo-FRT kanR neoR
  15. A006 FRT-gb2-cm-FRT cmR –
  16. A007 loxP-gb2-cm-loxP cmR –
  17. A008 FRT-gb2-amp-FRT ampR –
  18. A009 loxP-gb2-amp-loxP ampR –
  19. A010 FRT-PGK-gb2-hygro-FRT hygR hygR
  20. A011 loxP-PGK-gb2-hygro-loxP hygR hygR

Recombinase Expression Plasmids

Optimized Cre and FLPe* expression plasmids for an efficient sitespecific recombination.
Prokaryotic expression plasmids Flpe and Cre
• For the removal of FRT or loxP flanked DNA
• Gene expression and plasmid propagation are tightly controlled
by temperature
• Available with various antibiotic resistance markers
• Compatible with ColE1 based plasmids (e.g. pUC, pBS or pBR322
derivative, RK2, R6K, cosmid, P1 and BAC)
*Flpe is a more thermostable derivative of wild type Flp displaying an enhanced
activity at 37°C (Buchholz et al. Nature Biotechnology, 16:657-662 (1998)).
Cat.No. Plasmid / Recombinase Selection

  1. A104 707-FLPe tetR
  2. A105 708-FLPe cmR
  3. A112 705-Cre cmR
  4. A113 706-Cre tetR

Eukaryotic expression plasmid for FLPe

• For the removal of FRT-flanked DNA, e.g. neomycin resistance
markers in mammalian cells.
Cat.No. Plasmid / Recombinase Selection
A201 pCAGGS-FLPe (Academia) puroR
, ampR
A202 pCAGGS-FLPe (Industry) puroR
, ampR

*Flpe is a more thermostable derivative of wild type Flp displaying an enhanced
activity at 37°C (Buchholz et al. Nature Biotechnology, 16:657-662 (1998)).
The use of this product is governed by the terms and conditions of the pCAGGSFLPe Material Transfer Agreement.
puro : Puromycin
CAGGS : Eukaryotic promoter
CI-578 : Prokaryotic promoter