GeneArt™ PerfectMatch TALs and GeneArt™ Precision TALs User
Contents
1. Scholze H and Boch J 2011 TAL effectors are remote controls for gene activation Curr Opin Microbiol 14 47 53 Smith D B Davern K M Board P G Tiu W U Garcia E G and Mitchell G F 1986 Mr 26 000 Antigen of Schistosoma japonicum Recognized by Resistant WEHI 129 J Mice is a Parasite Glutathione S transferase Proc Natl Acad Sci USA 83 8703 8707 32 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 33 34 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide For support visit thermofisher com support or email techsupportlalifetech com thermofisher com Thermo Fisher SCIENTIFIC 28 August 20152. SV40 nuclear localization signal NLS For nuclear localization Hax3 N terminus N terminus domain of the TAL DNA binding domain Allows targeting of the TAL effector to LRRK2 DNA sequences Hax3 C terminus C terminus domain of the TAL containing activation domain Fokl Fokl nuclease domain of the TAL TK polyA Allows selection of the plasmid in coli Ampicillin resistance gene Herpes Simplex Virus Thymidine Kinase TK polyadenylation signal allows efficient transcription termination and polyadenylation of mRNA Cole and Stacy 1985 attB1 and attB2 Sites for gateway adaption pUC origin Allows high copy number replication and growth in coli 28 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Map of pENTR gus Entry Vector Description Map of control vector pENTR gus is a 3841 bp entry vector containing the Arabidopsis thaliana gene for B glucuronidase gus Kertbundit et al 1991 and is included as a positive control with Gateway LR Clonase II Enzyme Mix Cat nos 11791 020 and 11791 100 The gus gene was amplified using PCR primers containing attB recombination sites The amplified PCR product was then used in a BP recombination reaction with pDONR 201 to generate the entry clone For more information about the BP recombination reaction refer to the Gateway Technology with Clonase II manual The figur
3. The region Vector map of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 23 Map of TAL MCS Entry Vector TAL MCS Entry The map below shows the elements of the TAL MCS Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone 24 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Multiple cloning site of TAL MCS Entry Vector Native TAL MCS 2849 2900 2951 3002 Truncated TAL MCS 1901 1952 2003 2054 2105 2156 The multiple cloning site for the Native TAL MCS entry clone is shown below The sequence of the TAL C terminus is in bold The MCS is underlined Restriction sites are labeled to indicate the cleavage site GAT CCT TTT GCC GGA ACA GCC GAT GAT TIC CCT GCC TTT AAT GAG GAA GAA Asp Pro Phe Ala Gly Thr Ala Asp Asp Phe Pro Ala Phe Asn Glu Glu Glu Pmel HindIII l l CTG GCC TGG CTG ATG GAA CTG CTG CCT CAG GGT TCC CGT TTA AAC AAG CTT Leu Ala Trp Leu Met Glu Leu Leu Pro Gln Gly Ser Arg Leu Asn Lys Leu EcoRI SacI Salil KpnI Clal Scal XhoI BamHI BglII l l l
4. from Step 7 previous page e Chemically competent E coli cells e S 0 C Medium warm to room temperature e pUC19 control use as a control for transformation if desired e LB plates containing 100 pg mL ampicillin two for each transformation warm at 37 C for 30 minutes e 42 C water bath e 37 C shaking and non shaking incubator Transformation 1 For each transformation aliquot 50 uL of chemically competent E coli cells procedure into a sterile microcentrifuge tube 2 Add 1 uL of the LR recombination reaction from Set up the LR recombination reaction Step 7 previous page into the tube containing 50 uL of competent cells and mix gently Do not mix by pipetting up and down Incubate on ice for 30 minutes Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 450 uL of room temperature S O C Medium Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour MOY Gl gt 9 8 Spread 20 uL and 100 uL from each transformation on a prewarmed selective plate and incubate overnight at 37 C We generally plate 2 different volumes to ensure that at least 1 plate has well spaced colonies 9 An efficient LR recombination reaction should produce gt 5000 colonies if the entire LR reaction is transformed and plated 14 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Analyze transformants Analyze positive clones Analyz
5. Val Val 911 ACAAGTTTE AAAAAAGC AGGCTN NAC C TTG TAC AAA GTG GTT TGTTCAAACA TGTTTTTTCG TCCGAN NTG GGT CGA AAG AAC ATG TTT CAC CAA attB1 attB2 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 11 Perform the LR recombination reaction Introduction Perform an LR recombination reaction between the entry clone and the appropriate destination vector We recommend that you include a positive control see below and a negative control no LR Clonase II Enzyme Mix in your experiment TM Note This step is not required when using the GeneArt PerfectMatch TAL N TAL FoklI CMV Positive control The pENTR gus plasmid is used as a positive control for LR recombination and expression Using the pENTR gus entry clone in an LR recombination reaction with a destination vector will allow you to generate an expression clone containing the gene encoding P glucuronidase gus The pENTR gus positive control is supplied with the LR Clonase TI Enzyme Mix TM TM LR Clonase II The LR Clonase IT Enzyme Mix is available separately The LR Clonase II Enzyme Mix Enzyme Mix combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer previously supplied as separate components in LR Clonase Enzyme Mix into a single tube format Use the protocol provided on page12 to TM perform the LR recombination reaction using LR Clonase II Enzyme Mix Note You may p
6. expression clones Y Analyze transformants for the presence of insert by restriction enzyme digestion or colony PCR Prepare purified plasmid DNA and transfect the cell line of choice 1 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 7 Create a TAL sequence GeneArt The following are guidelines and rules for generating the PerfectMatch TAL PerfectMatch TALs Sequence binding site rules The GeneArt PerfectMatch TALs offering allows the construction of TAL effector functional proteins directed to either 18 or 24 base DNA target sites e GeneArt PerfectMatch TALs are provided in two types of vectors 1 Gateway adapted entry vector Gateway adapted entry vectors allow easy transfer of target specific TAL domain through a LR recombination reaction into destination vectors designed to facilitate high level expression of the TAL effectors in your cell line of choice A Gateway adapted destination vector is needed for expression plasmid generation Choose a destination vector from our Gateway adapted vector portfolio 2 CMV expression vector mammalian expression vector The mammalian expression vector contains CMV promoter which drives high level expression of the TAL in mammalian systems PerfectMatch TALs provided in this vector can be directly used for expression in mammalian systems without the need for any intermediate sub cloning steps Optional
7. in your experiment to evaluate your results Plasmid Plasmid DNA for transfection in eukaryotic cells must be very clean and free preparation from contamination with phenol and sodium chloride Contaminants will kill the cells and salt may interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the S N A P MidiPrep Kit Cat no K1910 01 PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 or CsCl gradient centrifugation Positive control If you used the pENTR gus control vector in an LR recombination reaction with a destination vector you can use the resultant expression clone as a positive control for mammalian cell transfection and expression A successful transfection will result in B glucuronidase expression that can be detected by western blot or functional assay Methods of We recommend using Lipofectamine 3000 Reagent Catalog no L3000015 or transfection the transfection method recommended by the supplier of the cell type being used For more information refer to www thermofisher com transfection or contact Technical Support see page 31 16 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Appendix Map of N TAL Fokl Entry Vector N TAL Fokl Entry The map below shows the elements of the N TAL FokI Entry Vector The region of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you or
8. pg mL chloramphenicol IMPORTANT Do NOT use general E coli cloning strains including TOP10 or DH5a for propagation and maintenance of destination vectors as these strains are sensitive to the toxic effects of the ccdB gene 10 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Create an expression clone continued Gateway The LR reaction facilitates recombination of an attL substrate entry clone with an attR substrate destination vector to create an attB containing expression clone see diagram below This reaction is catalyzed by LR Clonase II Enzyme Mix recombination reactions attL attL attR attR attB attB attP attP LR Clonase II Destination lt gt Expression By product vector clone Recombination In the following example the recombination region of the expression clone resulting from the LR reaction between a TAL entry clone and the region of the de m dl Gateway pcDNA DEST40 destination vector sequence is shown expression clone Features of the recombination region e Shaded regions correspond to those DNA sequences transferred from the entry clone into the destination vector by recombination e Non shaded regions are derived from the destination vector e The underlined nucleotides flanking the shaded region correspond to bases 918 and 2601 respectively of the Gateway pcDNA DESTA40 destination vector sequence 918 2601 Pro Ala Phe Leu Tyr Lys
9. prophylactic purposes or d any sale resale leasing or licensing whether or not for research purposes Development Purpose means any a clinical activity following IND enabling preclinical toxicological studies or equivalents thereof b activity in any agricultural field trial including any such field trial that would be subject to regulation by the United States Department of Agriculture if the plants were to constitute genetically engineered organisms under 7 C F R 8 340 or any successor regulation c activity directed towards the submission of data generated using a Plant to the United States Department of Agriculture or any equivalent regulatory agency outside of the United States in support of an application for clearance approval or deregulation by such agency d scale up activities the primary focus of which is to increase from small scale to production scale or e any use of pigs cattle or sheep cells or pig cattle or sheep animals generated using this TALEN product for i agricultural food applications or ii animal models and biomaterials models used for research and development of therapeutic compounds or protocols and production of non cellular products or proteins for therapeutic cosmetic or neutraceutical applications Plant means any eukaryotic plants plant tissues or plant cells including green algae belonging to the plant kingdom defined as Viridiplantae including any progeny propagated th
10. 2 Pick 5 colonies and resuspend them individually in 48 uL of the PCR SuperMix remember to make a patch plate to preserve the colonies for further analysis Incubate reaction for 10 minutes at 94 C to lyse cells and inactivate nucleases Amplify for 20 to 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C Dye OM ES 209 Visualize by agarose gel electrophoresis The ccdB gene mutates at a very low frequency resulting in a very low number of false positives True expression clones will be ampicillin resistant and chloramphenicol sensitive Transformants containing a plasmid with a mutated ccdB gene will be ampicillin and chloramphenicol resistant To check your putative expression clone test for growth on LB plates containing 30 ng mL chloramphenicol A true expression clone will not grow in the presence of chloramphenicol GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 15 Transfection Introduction Once you have generated your expression clone you are ready to transfect the plasmid into the mammalian cell line of choice You may perform transient transfection experiments or use Geneticin selection to generate stable cell lines The neomycin resistance gene in pcDNA dest 40 Gateway vector allows for the selection of stable cell lines using Geneticin antibiotic We recommend that you include a positive control see below and a negative control mock transfection
11. 93 027 Lipofectamine 3000 Transfection Reagent 1 5 mL L3000015 59 11815 024 Kanamycin Sulfate 259 11815 032 Kanamycin Sulfate 100X liquid 100 mL 15160 054 1g 11811 023 as eee 59 11811 031 Geneticin Selective Antibiotic 20 mL 50 mg mL 10131 035 100 mL 50 mg mL 10131 027 PureLink HiPure Plasmid MiniPrep Kit 25 preps K2100 02 PureLink HiPure Plasmid MidiPrep Kit 25 preps K2100 04 TM A large selection of Gateway destination vectors are available to facilitate the expression of your gene of interest in virtually any protein expression system For more information about the vectors available refer to our website www thermofisher com or contact Technical Support page 31 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Documentation and support Customer and technical support Visit www thermofisher com support for the latest in services and support including e Worldwide contact telephone numbers e Product support including Product FAQs Software patches and updates e Order and web support e Product documentation including User guides manuals and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Quality Assurance The Quality Assurance Document QAD is a certificate of analysis that provides detailed quality control and product quali
12. PerfectMatch TAL cassette can be transferred directly into your expression vector of choice with the restriction enzymes Not I and Hind III e Each target site sequence is preceded by a 5 N PerfectMatch TAL protein allows binding to a DNA sequence preceded by any DNA base The letter N represents any base of A G C or T The 5 N does not count as one of the 18 or 24 bases to be selected for targeting your specific site e Design nuclease pairs with a spacing of 13 18 bp between the target sites on opposite strands of the DNA However we recommend a spacing of 15 16 bp between the target sites in order to achieve maximal nuclease activity The target sites can be either 18 or 24 bp in length Use the following image as a reference for the orientation of the binding domains Functional DNA binding domain domain NNNNNNNNNNNNNNNNNNN 15 16 bp _ 16b POVDMDOOODOD AUN e The contribution of individual binding motifs within the DNA binding domain to TAL effector binding efficiency is thought to differ since strong and weak binding motifs exist The A and T binding motifs are thought to fall within the weak binder category while the C and G binding motifs are thought to be strong binders Stretches of more than 5 weak binders should be avoided at the extreme 5 end of the binding domain not counting the 5 N or if they are not flanked by Cs It is recommended to select a TAL effector with a DNA binding domain c
13. USER GUIDE invitrogen GeneArt PerfectMatch TALs and GeneArt Precision TALs TAL effector expression system for genome editing Catalog Numbers 816508DE 816509DE 816510DE 816511DE 816512DE 816514DE 816516DE 816517DE 816518DE 816010DE 816011DE 816012DE Publication Number MANO0006670 Revision B 0 ThermoFisher SCIENTIFIC For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER TO THE EXTENT ALLOWED BY LAW LIFE TECHNOLOGIES AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT Limited Use Label License No 406 GeneArt Precision TAL Effector Products The purchase of this product conveys to the buyer limited non transferable rights under certain TAL Effector technology owned by inventors from Martin Luther Universitat Halle Wittenberg and licensed to Life Technologies Corporation to use this product and components of this product only to perform internal research for the sole benefit of the buyer The buyer may also use standard molecular biology techniques to make additional copies of this product for purposes of internal research for the sole benefit of the buyer but the buyer may not modify the sequence of the TAL Effector within this product The buyer cannot sell or otherwise transfer a thi
14. anisms made using this product or its components including but not limited to Plants in all cases hereunder to a third party or otherwise use this product its components or materials cells or organisms made using this product or its components for any Commercial Purpose or Development Purpose with the sole exception that buyer may transfer this product its components and or materials cells or organisms made using this product or its components to i the buyer s legal affiliates and or ii a scientific collaborator provided that each such legal affiliate and or scientific collaborator agrees in writing 1 not to transfer such product its components or materials cells or organisms made using such product or its components to any third party and 2 to use such product its components and materials cells and organisms made using such product and or its components solely for research as set forth in this limited use label license and not for Commercial Purposes or Development Purposes If this product is subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Commercial Purpose means any activity for consideration including but not limited to a any use directly or indirectly in manufacturing production production of biomolecules or quality control b any use to provide a service information or data for consideration c any use for therapeutic diagnostic or
15. ansfection Nature 337 387 388 Kertbundit S Greve H d Deboeck F Montagu M V and Hernalsteens J P 1991 In vivo Random P glucuronidase Gene Fusions in Arabidopsis thaliana Proc Natl Acad Sci USA 88 5212 5216 Lamb BM Mercer AC Barbas CF 2013 3rd Directed evolution of the TALE N terminal domain for recognition of all 5 bases Nucleic Acids Res 41 21 9779 9785 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Li T Huang S Zhao X Wright D A Carpenter S Spalding M H Weeks D P and Yang B 2011 Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes Nucleic Acids Res 39 6315 6325 Mak AN Bradley P Cernadas RA Bogdanove AJ Stoddard BL 2012 The crystal structure of TAL effector PthXo1 bound to its DNA target Science 335 716 719 Mussolino C and Cathomen T 2012 TALE nucleases tailored genome engineering made easy Curr Opin Biotechnol 23 5 644 650 Moscou M J and Bogdanove A J 2009 A simple cipher governs DNA recognition by TAL effectors Science 326 1501 Orosz A Boros I and Venetianer P 1991 Analysis of the Complex Transcription Termination Region of the Escherichia coli rrnB Gene Eur J Biochem 201 653 659 Ptashne M 1992 A Genetic Switch Phage Lambda and Higher Organisms Cambridge MA Cell Press
16. cids in length with two centrally located residues that make up a repeat variable domain RVD that dictates the affinity of the repeat for different nucleotide targets Combination and order of various repeat types define the genomic target site specificity of a particular TAL effector The deciphering of this TAL effector code led to the engineering of designer TAL effector proteins that function as a vehicle to target functionality of essentially any open region of the chromosomes of plants bacteria yeast flies and mammalian cells Boch et al 2009 Moscou and Bogdanove 2009 Activities such as activators repressors and nucleases have been demonstrated to be addressable via this powerful system Li et al 2011 Scholze and Boch 2011 Mussolino and Cathomen 2012 These tools have applications from efficient genomic editing and gene knock out for manipulating the chromosome to modulation of specific promoter activities to allow simple and complex metabolic manipulation in various species of cells The two versions of TALs available are GeneArt PerfectMatch TALs and GeneArt Precision TALs With GeneArt PerfectMatch TALs and GeneArt Precision TALs the researcher can determine the exact DNA loci they would like to have their functionality delivered to and have specific TAL genes built to perform the function The researcher will receive a Gateway adapted entry vector containing the coding sequence for a TAL nuclease or acti
17. d GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Map of TAL Fokl Entry Vector TAL Fokl Entry The map below shows the elements of the TAL FokI Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone 20 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Map of TAL vp16 Entry Vector TAL vp16 Entry The map below shows the elements of the TAL vp16 Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 21 Map of TAL vp64 Entry Vector TAL vp64 Entry The map below shows the elements of the TAL vp64 Entry Vector The region of Vector map the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone 22 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Map of TAL KRAB Entry Vector TAL KRAB Entry The map below shows the elements of the TAL KRAB Entry Vector
18. dered The complete sequence for your clone in gb format is available on the disk provided with your clone Vector map GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 17 Map of N TAL Fokl CMV Expression Vector N TAL Fokl CMV The map below shows the elements of the N TAL FokI CMV Expression Vector The region of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone Expression Vector map N TAL Fokl CMV expression vector term V5 18 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Features of GeneArt PerfectMatch TAL vectors Common N TAL Fokl Fokl CMV vector features FokL and N TAL FokI CMV The following elements are found in the GeneArt PerfectMatch TALs N TAL Feature Description V5 epitope Gly Lys Pro lle Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of the recombinant fusion protein by the Anti V5 antibodies Southern et al 1991 pUC origin Allows high copy number replication and growth in coli DNA binding domain Allows targeting of the TAL effector to specific DNA sequences DNA repeat variable domain TAL N term N terminus domain of the TAL containing translocation and nuclear localization signal tag It contains 3 amino acids muta
19. e transformants by PCR Confirm the expression clone 1 Pick 5 colonies and culture them overnight in LB or SOB medium containing 100 ug mL ampicillin 2 Isolate plasmid DNA using your method of choice We recommend using the PureLink HiPure Plasmid MiniPrep Kit Cat no K2100 02 or the PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 See Additional products p30 3 Analyze the plasmids by restriction analysis to confirm the presence of the insert You can also analyze positive transformants using PCR For PCR primers use a primer that hybridizes within the vector e g T7 Promoter Primer Catalog no N560 02 and one that hybridizes within your insert You will have to determine the amplification conditions If you are using this technique for the first time you may want to perform restriction analysis in parallel Artifacts may be obtained because of mispriming or contaminating template The protocol below is provided for your convenience Other protocols are suitable Materials needed PCR SuperMix High Fidelity Cat no 10790 020 Appropriate forward and reverse PCR primers 20 uM each Note To avoid PCR errors due to highly repetitive sequences we recommend designing primers that hybridize to the N terminal domain of the TAL sequence Procedure 1 For each sample aliquot 48 uL of PCR SuperMix High Fidelity into a 0 5 mL microcentrifuge tube Add 1 uL each of the forward and reverse PCR primer
20. e below summarizes the features of the peENTR gus vector The complete sequence for pENTR gus is available from our web site www thermofisher com lifescience or by contacting Technical Support see page31 3841 nucleotides affL1 bases 228 2039 attL2 bases 2014 2140 pUC origin bases 2200 2873 C Kanamycin resistance gene bases 2990 3804 C C complementary strand GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 29 Accessory products Introduction Additional products Gateway destination vectors 30 The products listed in this section may be used with GeneArt PerfectMatch TALs and GeneArt Precision TALs For more information refer to our website www thermofisher com or contact Technical Support see page 31 Many of the reagents suitable for use with the vectors are available separately Ordering information for these reagents is provided below For more information refer to our website www thermofisher com Item Quantity Catalog no Gateway LR Clonase II Enzyme Mix 20 reactions 11791 020 100 reactions 11791 100 Library Efficiency DH5 a Competent Cells 5x 0 2 mL 18263 012 One Shot TOP10 Chemically Competent 20 reactions C4040 03 E coli One Shot TOP10 Electrocompetent coli 20 reactions C4040 52 One Shot MAX Efficiency DH10B T1 Phage 20 reactions 12331 013 Resistant col Ampicillin 200 mg 115
21. erform the LR recombination reaction using LR Clonase Enzyme Mix if desired To use LR Clonase Enzyme Mix follow the protocol provided with the product Do not use the protocol for LR Clonase II Enzyme Mix provided in this manual as reaction conditions differ Materials needed You should have the following materials on hand before beginning e Purified plasmid DNA of your entry clone 50 150 ng L in TE pH 8 0 e Destination vector 150 ng L in TE pH 8 0 e LRClonase II Enzyme Mix Cat no 11791 020 11791 100 e TE Buffer pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA TM e Proteinase K solution supplied with the LR Clonase IT Enzyme Mix TM e pENTR gus positive control supplied with the LR Clonase II Enzyme Mix 12 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Perform the LR recombination reaction continued Set up the LR recombination reaction Follow this procedure to perform the LR reaction between your entry clone and the destination vector If you want to include a negative control set up a separate reaction but omit the LR Clonase II Enzyme Mix Add the following components to 1 5 mL microcentrifuge tubes at room temperature and mix Set up an additional set of reactions for your negative TM control You will not add LR Clonase II Enzyme Mix to these reactions Component Sample Positive control Entry clone 50 150 ng reaction 1 7 uL Destination
22. fication information for each product Document Certificates of Analysis are available on the disk provided with your clone Limited product Life Technologies Corporation and or its affiliate s warrant their products warranty as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 31 References Boch J Scholze H Schornack S Landgraf A Hahn S Kay S Lahaye T Nickstadt A and Bonas U 2009 Breaking the code of DNA binding specificity of TAL type III effectors Science 326 1509 1512 Boch J and Bonas U 2010 Xanthomonas AvrBs3 family type III effectors discovery and function Annu Rev Phytopathol 48 419 436 Bushman W Thompson J F Vargas L and Landy A 1985 Control of Directionality in Lambda Site Specific Recombination Science 230 906 911 Cole CN Stacy TP 1985 Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation Mol Cell Biol 5 8 2104 2113 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L and Ringold G M 1989 Cationic Liposome Mediated Tr
23. ion and growth in coli DNA binding domain Allows targeting of the TAL effector to specific DNA sequences DNA repeat variable domain TAL N term N terminus domain of the TAL containing translocation and nuclear localization signal tag NLS Truncated versions of the vector contain the SV40 nuclear localization signal NLS while native vectors contain the two endogenous NLS of the TAL TAL C term C terminus domain of the TAL containing activation domain C complementary strand TM Specific TAL entry The following features are found in the specific GeneArt Precision TALs entry vector features vector noted Vector Feature Description TAL Fokl Fokl Fokl nuclease domain of the TAL TAL vp16 vp64 vp16 or vp64 Effector domain of the TAL activator TAL KRAB KRAB repressor Effector domain of the TAL TAL MCS MCS Multiple cloning site for insertion of custom effector domains into the TAL TAL MCS Gly Ser linker Flexible peptide linker to prevent steric hindrance between domains GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 27 Specific TAL Fokl LRRK2 entry vector features Feature Description EF1alpha promoter Allows efficient high level expression of TAL Fokl protein V5 epitope tag Allows detection of the recombinant fusion protein by the Anti V5 The following features are found in the TAL FokI LRRK2 entry vector antibodies Southern et al 1991
24. l l l l GTC GAC GGT ACC GAA TTC ATC GAT AGT ACT CTC GAG GGA TCC GAG CTC AAG Val Asp Gly Thr Glu Phe Leu Ile Asp Ser Thr Leu Glu Gly Ser Glu Leu ATC TAG CTA AGT AGA CCC AGC TTT CTT GTA CAA AGT TGG CAT TAT AAG Lys The multiple cloning site for the Truncated TAL MCS entry clone is shown below The sequence of the TAL C terminus is in bold The MCS is underlined Restriction sites are labeled to indicate the cleavage site TTT TTT CAG TGT CAC TCT CAC CCT GCC CAG GCC TTT GAT GAT GCC ATG ACA Phe Phe Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp Ala Met Thr CAG TTT GGC ATG AGC AGA CAC GGA CTG CTG CAG CTG TTT AGA AGA GTG GGA Gln Phe Gly Met Ser Arg His Gly Leu Leu Gln Leu Phe Arg Arg Val Gly GTG ACA GAA CTG GAG GCC AGA TCC GGA ACC CTG CCT CCT GCC TCT CAG AGA Val Thr Glu Leu Glu Ala Arg Ser Gly Thr Leu Pro Pro Ala Ser Gln Arg Pmel HindIII Sail KpnI l l l TGG GAT AGG ATT CTG CAG GGT TCC CGT TTA AAC AAG CTT GTC GAC GGT ACC Trp Asp Arg Ile Leu Gln Gly Ser Arg Leu Asn Lys Leu Val Asp Gly Thr SacI EcoRI Clal Scal XhoI BamHI BglII l l l l l GAA TTC ATC GAT AGT ACT CTC GAG GGA TCC GAG CTC AAG ATC TTA GCT AAG Glu Phe Leu Ile Asp Ser Thr Leu Glu Gly Ser Glu Leu Lys TAG ACC CAG CTT TCT TGT ACA AAG TTG GCA TTA TAA GAA GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 25 Map of TAL Fokl LRRK2 Entry Vector TAL Fokl LRRK2 The map below shows the elements of the TAL FokI LRRK2 Entry Vector The Entry Vector map regi
25. lly excluded from this definition For information on obtaining additional rights to TAL Effector technology for any use not permitted herein except use in Plants please contact Life Technologies at outlicensing dlifetech com For information on obtaining additional rights to TAL Effector technology for any use in Plants not permitted herein please contact Two Blades Foundation at info a 2blades org or Two Blades Foundation 1630 Chicago Avenue Suite 1907 Evanston IL 60201 USA For information on donating mice to The Jackson Laboratory please visit their website at http www jax org grc index html Limited Use Label License No 521 GeneArt Precision TALEN Products The purchase of this product conveys to the buyer limited non transferable rights under certain TALEN technology owned and or controlled by Cellectis SA including patents owned by the University of Minnesota and lowa State University and licensed to Life Technologies Corporation to use this product and components of this product only to perform internal research for the sole benefit of the buyer The buyer may also use standard molecular biology techniques to make additional copies of this product for purposes of internal research for the sole benefit of the buyer except the buyer may not modify the sequence of the TAL effector within this TALEN product The buyer cannot sell or otherwise transfer a this product b its components or c materials cells or org
26. n including but not limited to a any use directly or indirectly in manufacturing production production of biomolecules or quality control b any use to provide a service information or data for consideration c any use for therapeutic diagnostic or prophylactic purposes or d any sale resale leasing or licensing whether or not for research purposes Development Purpose means any a clinical activity following IND enabling preclinical toxicological studies or equivalents thereof b activity in any agricultural field trial including any such field trial that would be subject to regulation by the United States Department of Agriculture if the plants were to constitute genetically engineered organisms under 7 C F R 8 340 or any successor regulation c activity directed towards the submission of data to the United States Department of Agriculture or any equivalent regulatory agency outside of the United States in support of an application for clearance approval or deregulation by such agency or d scale up activities the primary focus of which is to increase from small scale to production scale Plant means any eukaryotic plants plant tissues or plant cells including green algae belonging to the plant kingdom defined as Viridiplantae including any progeny propagated through any number of generations or unmodified derivatives of such plants tissues or cells For clarity fungi or blue green algae are specifica
27. neArt Precision TALs User Guide 1 Product information Product description Introduction Invitrogen GeneArt PerfectMatch TALs and GeneArt Precision TALs are optimized to deliver transcriptional effectors to cells in a sequence specific manner These TALs are provided as Gateway adapted entry vectors The sequence is transferred from the entry vector to a destination vector by LR recombination resulting in high level expression of the TAL effectors The GeneArt PerfectMatch TALs are also provided as a CMV expression vector for high level expression in mammalian cells without an LR recombination step Kit contents and storage Ordering information Contents Shipping storage GeneArt PerfectMatch TALs functional domain Catalog no Truncated N TAL Fokl 816508DE Truncated N TAL Fokl CMV 816509DE GeneArt Precision TALs functional domain Catalog no Native TAL Fokl 816510DE Truncated TAL Fokl 816511DE Native TAL vp16 activator 816512DE Native TAL vp64 activator 816514DE Native TAL MCS 816516DE Truncated TAL MCS 816517DE Native TAL KRAB repressor 816518DE Validated GeneArt Precision TAL Catalog no Truncated TAL Fokl LRRK2 specific 816012DE DNA binding domain Catalog no 18 Nucleotide Binding Domain containing your specific TAL 816010DE 24 Nucleotide Binding Domain containing your specific TAL 816011DE 5 ug of vector DNA lyophilized The TAL vecto
28. omosome and the switch between the lytic and lysogenic pathways Ptashne 1992 In Gateway Technology the components of the lambda recombination system are modified to improve the specificity and efficiency of the system Bushman et al 1985 LR recombination An LR recombination reaction is performed between the entry clone and the reaction destination vector of choice to generate an expression clone The LR recombination reaction is mediated by LR Clonase II Enzyme Mix a mixture of the bacteriophage A Integrase Int and Excisionase Xis proteins and the E coli Integration Host Factor IHF protein 6 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Methods Experimental outline TM Experimental The table below outlines the steps required to express your GeneArt outline steps PerfectMatch TALs and GeneArt Precision TAL s in cells Step Action Page Determine the sequence of the binding site for your 1 TAL effector protein Synthesize TAL sequence and clone into a Gateway adapted entry vector of choice to generate an entry clone Or clone TAL sequence into the CMV expression vector Perform an LR recombination reaction by mixing the entry clone and the appropriate destination vector with Gateway LR Clonase II Enzyme Mix 12 Note This step is not required with the CMV vector Transform the recombination reaction into competent E coli cells and select for
29. omposed of mixed binding motifs for best results 8 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide e Inthe context of the living cell DNA accessibility also determines TAL effector efficiency Chromatin structure DNA methylation and or proteins bound to the DNA may interfere with TAL binding GeneArt The following are guidelines and rules for generating the Precision TAL Precision TALs sequence binding site rules e The GeneArt Precision TALs offering allows the construction of TAL effector functional proteins directed to either 18 or 24 base DNA target sites e Fach target site must be preceded by a 5 T because the N terminus of the TAL effector protein contains a conserved T binding motif The 5 T does not count as one of the 18 or 24 bases to be selected for targeting your specific site e Nuclease pairs need to be designed with a spacing of 13 18 bp between the target sites on opposite strands of the DNA Both target sites must be preceded by a5 T The target sites can be either 18 or 24 bp in length The following image should be used as a reference for the orientation of the binding domains Functional DNA binding domain domain TNNNNNNNNNNNNNNNNON 13 18 bp gt cI TETTE e The contribution of individual binding motifs within the DNA binding domain to TAL effector binding efficiency is thought to differ since strong and weak binding motifs exist The A and T binding motifs are
30. on of the entry clone corresponding to the TAL is variable depending upon the length of the sequence you ordered The complete sequence for your clone in gb format is available on the disk provided with your clone V5 NLS TAL N Term LRRK F repeats Or LRRK R repeats Fokl TAL C term Tk polyA 26 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Features of GeneArt Precision TALs entry vectors Common TAL entry The following elements are found in the GeneArt Precision TALs entry vectors TAL FokI TAL vp16 vp64 TAL MCS All features have been functionally tested These features do not apply to the TAL FolkI LRRK2 entry vector vector features Feature Description rrnB T1 and T2 transcription Protects the cloned gene from expression by vector encoded terminators promoters thereby reducing possible toxicity Orosz et al 1991 M13 Forward 20 priming site Allows sequencing in the sense orientation M13 Reverse C priming site Allows sequencing in the antisense orientation attL1 and attL2 sites Allows recombinational cloning of the gene of interest from an entry clone Landy 1989 Kanamycin resistance gene Allows selection of the plasmid in coli V5 epitope Allows detection of the recombinant fusion protein by the Anti V5 Gly Lys Pro lle Pro Asn Pro antibodies Southern et al 1991 Leu Leu Gly Leu Asp Ser Thr pUC origin Allows high copy number replicat
31. ooooccccnnnncinocononononnnnnnnnnononccnnnnnnnnnnn nn nn nn cnn nan nnnn AEEA EEEn nnne EEEn 12 Transform competent E coli cells cccccceeeecceceececeeeeececeacaeeeeeeececcaaeeeeeeeeeseceaaeceeeeeeeseceaaeeeeeeeeeeennaees 14 AnalyZetranStorm arts 5 ccneescenics yeeteh A io 15 Transfection 2 Sib tdt A tddi 16 PAP PON iia iaa cda 17 Map of N TAL Fokl Entry Vector ccoo ddd a A A a SA 17 Map of N TAL Fokl CMV Expression Vector cccccceceeeeeeeeneceeeeeeeeecanaeceeeeeeesecencaeceeeeeeesensiaeeseees 18 Features of GeneArt PerfectMatch TAL Vectors cccsccscscsscsessssescessessevssetstssevsesesstsesevstssetsesneesess 19 Map ot TAL FokliEntry Vector comica 20 Map of TAL vp16 Entry Vector eaa a a r aera aa ae aaa aa aaa a aeii 21 Map ot TAL vpo4 Entry Vector eto a Udo 22 Map of TAL KRAB Entry Vector iii Aa 23 Map of TAL MCS Entry Vector aradaki niar d did ed 24 Multiple cloning site of TAL MCS Entry VectOF ooococonncoccnnnococononocancnnnononcnnnnnn nn crono cnn rra n nar nnnn rra 25 Map of TAL Fokl LRRK2 Entry Vectores or reenn AEE EA cnn nano rca rro nn rre rara 26 Features of GeneArt Precision TALS O 225 55 ceiver n aa tu csane dace a ae E E RAA EEA 27 Map of pENTR gus EMF VECON ati A A A AA ERA 29 SA ANS 30 DocCumentation and TS AAA NT 31 MER SS A 31 References ici 32 About this guide Revision history IEA BO August 2015 Add LRRK2 TAL vector information GeneArt PerfectMatch TALs and Ge
32. r contains a functional domain and an 18 or 24 nucleotide DNA binding domain All GeneArt PerfectMatch TALs and GeneArt Precision TALs are shipped at room temperature Do not store lyophilized DNA for a prolonged time Upon receipt resuspend the vector and store at 20 C GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide Resuspend the vector DNA Add 50 pL of distilled water or 10 mM Tris HCl pH 8 0 to the tube containing the vector and incubate for one hour at room temperature Resuspend the vector DNA by gently pipetting up and down 5 10 times Store the resuspended vector DNA at 20 C Antibiotic resistance markers are indicated on each tube label The standard delivery amount of DNA is 5 ug Contents description TAL effectors GeneArt PerfectMatch TALs and GeneArt Precision TALs Transcription activator like TAL effector proteins are naturally occurring transcriptional activators secreted by Xanthamonas spp into their plant hosts They are injected into plant host cells via a Type III secretion system and travel to the nucleus where they bind to and activate specific promoter sequences that lead to changes that are permissive for bacterial infection Boch and Bonas 2010 TAL effector proteins consist of constant N and C terminal domains containing translocation and nuclear localization activation signals respectively flanking a central repeat domain Each repeat is 34 35 amino a
33. r of choice We recommend that you review this section and the next section entitled Perform the LR recombination reaction pages 12 13 before proceeding TM Note This step is not required when using the GeneArt PerfectMatch TAL N TAL FokI CMV Resuspend the Each destination vector is supplied as 6 ug of lyophilized plasmid To use resuspend the destination plasmid in 40 pL of TE Buffer pH 8 0 to a final concentration of 150 ng pL vectors Note Destination vectors are supplied as supercoiled plasmids The linearization of the destination vector is NOT required to obtain optimal results for any downstream application Propagate the Entry clone vectors Propagate and maintain your entry clone using a recA endA E coli strains like TOP10 TOP10F DH5a JM109 or equivalent for transformation Select transformants on LB plates containing 50 100 pg mL kanamycin Prepare a glycerol stock of each plasmid for long term storage Destination vector If you wish to propagate and maintain your destination vectors prior to recombination we recommend using One Shot ccdB Survival T1 Chemically Competent E coli Cat no C7510 03 for transformation The One Shot ccdB Survival T1 E coli strain is resistant to the toxic effects of the ccdB gene and can support the propagation of plasmids containing the ccdB gene To maintain the integrity of the vector select for transformants in media containing 50 100 pg mL ampicillin and 15 30
34. rough any number of generations or unmodified derivatives of such plants tissues or cells For clarity fungi or blue green algae are specifically excluded from this definition For information on obtaining rights to TALEN technology for any use not permitted herein except use in the therapeutic field and in Plants please contact Life Technologies at outlicensinglalifetech com For information on obtaining rights to TALEN technology for any use in the therapeutic field please contact Cellectis SA at business development dcellectis com For information on obtaining rights to TALEN technology for any use in Plants not permitted herein please contact Cellectis Plant Sciences Inc at businessdevelopment_cps dcellectis com 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified TALEN is a trademark of Cellectis Contents ADOUE this g der ear aaee ti da ii lid deis 1 REVISION MAO riar aaa di E TR se deca dic aiii 1 Product InTOrMatiON coi iaa 2 Products A A a aaa A aaa 2 Kit contents and storage ii a dd 2 Contents deScripliO Martirio airada Sed ade se 3 Methods te wives a a rA a a aa e aaa E T e a aa raa te ensues di a aa a E aE aean reaa aee TEA reena suusvandasentvd cvesuenee 7 Experimental outlines nns ci A ee Ea 7 Create a TAE Seguente h ai a a lada 8 Create an ES 10 Perform the LR recombination reactiON oo
35. s product b its components or c materials cells or organisms made using this product or its components including but not limited to Plants in all cases hereunder to a third party or otherwise use this product its components or materials cells or organisms made using this product or its components for any Commercial Purpose or Development Purpose with the sole exception that buyer may transfer this product its components and or materials cells or organisms made using this product or its components to i the buyer s legal affiliates and or ii a scientific collaborator provided that each such legal affiliate and or scientific collaborator agrees in writing 1 not to transfer such product its components or materials cells or organisms made using such product or its components to any third party and 2 to use such product its components and materials cells and organisms made using such product and or its components solely for research as set forth in this limited use label license and not for Commercial Purposes or Development Purposes If this product is subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Mice generated using this product may be donated to The Jackson Laboratory Please include a copy of this limited use label license with all donations to identify the TAL Effector technology used Commercial Purpose means any activity for consideratio
36. ted from T TALs NLS Truncated versions of the vector contain the SV40 nuclear localization signal NLS TAL C term C terminus domain of the TAL containing activation domain Fokl Fokl nuclease domain of the TAL Specific N TAL Fokl Fokl CMV vector features vector noted TM The following features are found in the specific GeneArt PerfectMatch TAL Vector Feature Description N TAL Fokl rrnB T1 and T2 transcription Protects the cloned gene from expression by terminators vector encoded promoters thereby reducing possible toxicity Orosz et al 1991 N TAL Fokl M13 Forward 20 priming site Allows sequencing in the sense orientation N TAL Fokl attL1 and attL2 sites Allows recombinational cloning of the gene of interest from an entry clone Landy 1989 N TAL Fokl Kanamycin resistance gene Allows selection of the plasmid in coli N TAL Fokl CMV Pemv Human cytomegalovirus CMV immediate early promoter enhancer BGHpA Bovine growth hormone BGH polyadenylation signal N TAL Fokl CMV Allows efficient high level expression of TAL Fokl protein Allows efficient transcription termination and polyadenylation of mRNA N TAL Fokl CMV Ampicillin resistance gene C Allows selection of the plasmid in coli N TAL Fokl CMV T7 promoter priming site Allows n vitro transcription in the sense orientation and sequencing through the insert C complementary stran
37. thought to fall within the weak binder category while the C and G binding motifs are thought to be strong binders Stretches of more than 5 weak binders should be avoided at the extreme 5 end of the binding domain not counting the 5 T or if they are not flanked by Cs It is recommended to select a TAL effector with a DNA binding domain composed of mixed binding motifs for best results e In the context of the living cell DNA accessibility also determines TAL effector efficiency It is possible that chromatin DNA methylation and or proteins bound to the DNA may interfere with TAL binding e Although promoter structure varies and specific rules regarding design are currently lacking it is recommended that TAL transcription factors used for transcriptional activation of natural promoters be positioned upstream of the TATA box or in some cases downstream of the transcriptional start site Selecting a target site directly over the TATA box or other known transcription factor binding site is not recommended Be sure that the natural ATG is present and that no premature ATG which may interfere with the natural translational start is transcribed GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 9 Create an expression clone Introduction To create an expression clone perform the LR recombination reaction to transfer the gene of interest from the Gateway adapted entry vector into your destination vecto
38. vator designed to bind a specific 18 or 24 base DNA sequence of choice The GeneArt PerfectMatch TALs are also available in a CMV expression vector GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 3 GeneArt GeneArt PerfectMatch TALs can be designed to target any locus in the genome PerfectMatch TALs since there are no restrictions for the 5 base Previously target sites for customized TAL effectors required a 5 T in the target sequences for maximal binding activities The 5 T constraint limited the flexibility of TAL effector target sites in the genome and prevented some specific sites in the genome from being targeted Structure studies suggested the N terminal domain NTD of the TAL effectors not the central repeat domain is responsible for the interaction with the 5 T of the target We developed our second generation TALs GeneArt PerfectMatch TALs by mutating the N terminal domain to reduce its specificity for 5 T GeneArt PerfectMatch TALs can target DNA sequences with any 5 base T G C or A with performance comparable to that of GeneArt Precision TALs N TAL Fokl and GeneArt PerfectMatch TALs contain a truncated TAL engineered with Fokl N TAL Fokl CMV nuclease The FokI TAL nuclease pair binds to duplex DNA at the target sites designated by the DNA binding domains to cleave the DNA There are two versions of GeneArt PerfectMatch TALs e N TAL FokI a Gateway adapted entr
39. vector 150 ng uL Tul 1 uL pENTR gus 50 ng pL 2 uL TE Buffer pH 8 0 to8 uL 5 uL TM Remove the LR Clonase IT Enzyme Mix from 20 C and thaw on ice 2 minutes Vortex the LR Clonase II Enzyme Mix briefly twice 2 seconds each time Add 2 uL of LR Clonase II Enzyme Mix to each sample or positive control reaction listed above Mix well by pipetting up and down Do not add LR Clonase II Enzyme Mix to negative control reactions Reminder Return LR Clonase II Enzyme Mix to 20 C immediately after use Incubate reactions at 25 C for 1 hour Note For most applications 1 hour will yield a sufficient number of colonies for analysis Depending on your needs the length of the recombination reaction can be extended up to 18 hours For large plasmids 210 kb longer incubation times will yield more colonies Add 1 uL of Proteinase K solution to each reaction Incubate for 10 minutes at 37 C Proceed to Transform competent E coli cells next page Note You may store the LR reaction at 20 C for up to 1 week before transformation if desired GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 13 Transform competent coli cells Introduction Once you have performed the LR recombination reaction transform chemically competent E coli with the resulting expression clone Materials needed You should have the following materials on hand before beginning e LR recombination reaction
40. x DNA at the target sites designated by the DNA binding domains to cleave the DNA Functional DNA binding domain domain TNNNNNNNNNNNNNNNNNN POWOWOWOYOYN ci TEENE NNNNNNNNNNNNNNNNNNT TAL VP16 and GeneArt Precision TALs engineered with the VP16 or VP64 activators can be TAL VP64 used to increase the expression level of endogenous or recombinant genes VP16 is a trans acting protein originating from the herpes simplex virus that forms a complex with host transcription factors to induce immediate early gene transcription VP64 is a tetrameric form of the VP16 minimal activation domain Functional DNA binding domain domain TNNNNNNNNNNNNNNNNNN DOTON TAL KRAB GeneArt Precision TALs engineered with the KRAB repressor can be used to down regulate the expression level of endogenous or recombinant genes Functional DNA binding domain domain TNNNNNNNNNNNNNNNNNN POODOOUL GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide 5 TAL MCS GeneArt Precision TALs that include a multiple cloning site MCS allow the user to clone any desired effector domain and target the protein to any locus within the genome Functional DNA binding domain domain A TNNNNNNNNNNNNNNNNNN POWOWOWE Gateway The Gateway Technology is a cloning method based on the bacteriophage Technology lambda site specific recombination system which facilitates the integration of lambda into the E coli chr
41. y vector which allows easy transfer through a LR recombination reaction to destination vectors designed to facilitate high level expression of the TAL effectors in your cells of choice e N TAL FokI CMV a CMV expression vector which contains a CMV promoter to drive high level expression of the TAL in mammalian systems It can be directly used without extra subcloning Functional DNA binding domain domain NNNNNNNNNNNNNNNNNNN POWOWOWOD OY NNNNNNNNNNNNNNNNNNN 4 GeneArt PerfectMatch TALs and GeneArt Precision TALs User Guide GeneArt Unlike GeneArt PerfectMatch TALs Precision TALs have a conserved T binding motif at the N terminus of the TAL effector protein and so require a 5 T for maximal binding activity GeneArt Precision TALs are available as the following Gateway adapted entry vectors TAL Fokl TAL FokI LRRK2 validated TAL VP16 TAL VP64 TAL KRAB and TAL MCS Precision TALs TAL Fokl and GeneArt Precision TALs engineered with the FokI nuclease can be used for Validated TAL Foki targeting specific genes for silencing GeneArt Precision TAL LRRK2 vectors have been designed and validated for silencing the LRRK2 gene by FokI nuclease Note see References for validation details Fok is a type IIS restriction endonuclease from Flavobacterium okeanokoites consisting of an N terminal DNA binding domain and a non specific DNA cleavage domain at the C terminal A FokI nuclease pair binds to duple
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