Concert 96 Protein Screen - Thermo Fisher Scientific
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1. Technologies grants you a non exclusive sub license under patents assigned to BSA for the use of this technology including the enclosed materials based upon the following conditions 1 these materials are to be used for non commercial research purposes only A separate license under patents owned by BSA is required for any commercial use including the use of these materials for research purposes or production purposes by any commercial entity Information about commercial license may be obtained from The Office of Technology Transfer Brookhaven National Laboratory Bldg 475D P O Box 5000 Upton New York 11973 5000 Phone 516 344 7134 2 No materials that contain the cloned copy of the T7 gene 1 the gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this sub license and agrees to be bound by its terms This limitation applies to strains BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE CE6 BL21 SI Competent Cells and any derivatives that are made of them You may refuse this sub license by returning this product unused in which case Life Technologies accept return of the product with a full refund By keeping or using this product you agree to be bound by the terms of this license This product is the subject of one or more of U S Patent Numbers 5 910 438 6 180 407 and 7 176 029 and corresponding foreign patents and is sold under license from th2. located between the two attR sites for counterselection e The ccdB gene located between the two attR sites for negative selection e V5 epitope and 6xHis tag for detection and purification optional e Ampicillin resistance gene for selection in E coli e pBR322 origin for low copy replication and maintenance of the plasmid in E coli e acl gene encoding the lac repressor to reduce basal transcription from the T7lac promoter For a map of pET DEST42 see page 9 continued on next page Overview continued The Gateway Technology T7 Regulated Expression T7lac Promoter Gateway is a universal cloning technology that takes advantage of the site specific recombination properties of bacteriophage lambda Landy 1989 to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems To express your gene of interest using Gateway cloning technology simply 1 Clone your gene of interest into a Gateway entry vector to create an entry clone 2 Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway destination vector e g pET DEST42 3 Transform your expression clone into a BL21 strain of choice Induce expression of your protein with IPTG For more information on the Gateway System refer to the Gateway Technology Manual This manual is available for downloading from our Web site www invitrogen com or
3. 500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Safety Data Sheets SDSs are available at www invitrogen com sds Certificate of The Certificate of Analysis provides detailed quality control and Analysis product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box continued on next page 11 Technical Service continued Limited Warranty 12 Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instruction
4. GAG GAG CCA GAG CTA AGA TGC 6xHis tag Arg Thr Gly His His 2168 CGT ACC GGT CAT CAT GCA TGG CCA GTA GTA The Next Step His His His His ax CAC CAT CAC CAT TGA GTTTGATCCG GCTGCTAACA GTG GTA GTG GTA ACT CAAACTAGGC CGACGATTGT Once you have generated your expression clone you will need to transform it into a BL21 E coli strain for expression studies Proceed to the next section for guidelines on choosing a BL21 host strain BL21 E coli Strains Introduction This section provides information on strains available from Invitrogen that are specifically designed for use with T7 promoter based expression systems Note Other BL21 strains are suitable Recommended See the table below for recommended strains and their Strains benefits For more information on these strains and other BL21 strains available from Invitrogen refer to our Web site www invitrogen com or contact Technical Service page 11 Product Benefit Catalog no BL21 Star DE3 One Shot Mutation in RNaseE improves C6010 03 Chemically Competent E coli stability of mRNA transcripts and increases protein yields BL21 AI One Shot T7 RNA polymerase under the C6070 03 Chemically Competent E coli control of araBAD promoter for inducible expression and low basal levels Especially useful for expression of toxic genes Expression and Analysis Introduction Basic Strategy Plasmid Preparation Ch
5. ation to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept retur
6. by contacting Technical Service page 11 pET DEST42 uses elements from bacteriophage T7 to control expression of heterologous genes in E coli In the vector expression of the gene of interest is controlled by a strong bacteriophage T7 promoter that has been modified to contain a lac operator sequence see below In bacteriophage T7 the T7 promoter drives expression of gene 10 10 T7 RNA polymerase specifically recognizes this promoter To express the gene of interest it is necessary to deliver T7 RNA polymerase to the cells by inducing expression of the polymerase or infecting the cell with phage expressing the polymerase pET DEST42 has been designed to contain a T7lac promoter to drive expression of the gene of interest The T7lac promoter consists of a lac operator sequence placed downstream of the T7 promoter The Jac operator serves as a binding site for the lac repressor encoded by the lacl gene and functions to further repress T7 RNA polymerase induced basal transcription of the gene of interest in BL21 strains Using pET DEST42 Important Propagating pET DEST42 Entry Clone The pET DEST42 vector is supplied as a supercoiled plasmid Although Invitrogen has previously recommended using a linearized destination vector for more efficient recombination further testing has found that linearization of this vector is NOT required to obtain optimal results for any downstream application If you wish to p
7. e Universit Libre de Bruxelles for research purposes only ccdB selection technology is described in Bernard et al Positive Selection Vectors Using the F Plasmid ccdB Killer Gene Gene 148 1994 71 74 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity For licensing information for use in other than research please contact Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail References Gold L 1988 Posttranscriptional Regulatory Mechanisms in Escherichia coli Ann Rev Biochem 57 199 233 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Annu Rev Biochem 58 913 949 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Miller J H 1992 A Short Course in Bacterial Genetics A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria Plainview New York Cold Spring Harbor Laboratory Press Rosenberg A H Lade B N Chui D S Lin S W Dunn J J and Stud
8. encodes a protein called B lactamase This protein is secreted into the medium where it hydrolyzes ampicillin inactivating the antibiotic Since B lactamase is catalytic ampicillin is rapidly removed from the medium resulting in non selective conditions If your plasmid is unstable this may result in the loss of plasmid and low expression levels continued on next page Expression and Analysis continued Using Carbenicillin Detection of Recombinant Fusion Proteins Note Purification of Recombinant Fusion Proteins Carbenicillin is generally more stable than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help to increase expression levels by preventing loss of the pET DEST42 expression plasmid If you wish to use carbenicillin perform your transformation and expression experiments in LB containing 50 pg ml carbenicillin Note If your gene of interest is highly toxic increasing the concentration of carbenicillin used from 50 pg ml to 200 ug ml may help to increase expression levels To detect expression of your recombinant fusion protein by Western blot analysis you may use antibodies against the appropriate epitope see page v for ordering information or an antibody to your protein of interest In addition the Positope Control Protein Catalog no R900 50 is available from Invitrogen for use as a positive control for detection of fusion proteins containing a V5 ep
9. enicillin 5g 10177 012 IPTG isopropylthio B galactoside 1g 15529 019 Detection of Recombinant Expression of your recombinant fusion protein can be detected using an antibody to the appropriate epitope Proteins Horseradish peroxidase HRP or alkaline phosphatase AP conjugated antibodies allow one step detection using colorimetric or chemiluminescent detection methods The amount of antibody supplied is sufficient for 25 Western blots Product Epitope Catalog no Anti V5 Antibody Detects 14 amino acid epitope R960 25 Anti V5 HRP Antibody derived IF R961 25 proteins of the paramyxovirus Anti V5 AP Antibody SV5 Southern et al 1991 R962 25 GKPIPNPLLGLDST Anti His C term Antibody Detects the C terminal R930 25 polyhistidine 6xHis tag requires Anti His C term HRP the free carboxyl group for R931 25 Antibody detection Lindner et al 1997 Anti His C term AP HHHHHH COOH R932 25 Antibody continued on next page Accessory Products continued Purification of If your gene of interest is in frame with the C terminal Recombinant peptide containing the V5 epitope and the polyhistidine Fusion Protein 6xHis tag you may use Immobilized Metal Affinity Chromatography IMAC to purify your recombinant fusion protein The ProBond Purification System or bulk ProBond resin are available separately from Invitrogen See the table below for order
10. ge T7 which allows efficient transcription termination bla promoter Allows expression of the ampicillin resistance gene Ampicillin resistance gene Allows selection of the plasmid in E coli pBR322 origin Allows replication and maintenance in E coli lacl ORF Encodes lac repressor which binds to the T7lac promoter to block basal transcription of the gene of interest Also binds the lacUV5 promoter in BL21 strains containing the ADE3 lysogen to repress transcription of T7 RNA polymerase 10 Technical Service Web Visit the Invitrogen Web site at www invitrogen com for Resources Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1760 602 6
11. ier F W 1987 Vectors for Selective Expression of Cloned DNAs by T7 RNA Polymerase Gene 56 125 135 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 Studier F W and Moffatt B A 1986 Use of Bacteriophage T7 RNA Polymerase to Direct Selective High Level Expression of Cloned Genes J Mol Biol 189 113 130 Studier F W Rosenberg A H Dunn J J and Dubendorff J W 1990 Use of T7 RNA Polymerase to Direct Expression of Cloned Genes Meth Enzymol 185 60 89 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 15 Notes invitrogen by Lite technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
12. ing information Product Quantity Catalog no ProBond Nickel chelating Resin 50 ml R801 01 150 ml R801 15 ProBond Purification System 6 purifications K850 01 ProBond Purification System with 1 kit K853 01 Anti His C term HRP Antibody ProBond Purification System with 1 kit K854 01 Anti V5 HRP Antibody Purification Columns 50 R640 50 10 ml polypropylene columns vi Overview Description The pET Expression System Features Methods pET DESTA2 is a 7 4 kb vector adapted for use with the Gateway Technology It is designed to allow high level inducible expression of recombinant fusion proteins in E coli using the pET system The pET system was originally developed by Studier and colleagues and takes advantage of the high activity and specificity of the bacteriophage T7 RNA polymerase to allow regulated expression of heterologous genes in E coli from the T7 promoter Rosenberg et al 1987 Studier and Moffatt 1986 Studier et al 1990 For more information about T7 regulated expression see the next page pET DEST42 contains the following elements e T7lac promoter for high level IPTG inducible expression of the gene of interest in E coli see next page for more information e Two recombination sites attR1 and attR2 downstream of the T7 promoter for recombinational cloning of the gene of interest from an entry clone e Chloramphenicol resistance gene
13. invitrogen by technologies pET DEST42 Gateway Vector A destination vector for high level inducible expression in E coli Catalog no 12276 010 Rev Date 18 June 2010 Manual part no 25 0519 MAN0000280 Table of Contents Important Information r un u eseneennenemensanasiennn iv Accessory Prod Uicts Murias v Method Si isaisa esea anai aaaea saraaa aaia kaap Ada aa aaar aaa 1 O 1 Using PET DEST42 iii asian alu 3 BL21 E coli Strains ui ee no 6 Expression atid Analysis cita 7 A A ae tides na pet seeneheen 9 Map and Features of PET DEST oerrinne 9 Technical Service nicotina tesi 11 Purchaser Notification cui dois 13 A nnan teste aaa E orient 15 Important Information Shipping and pET DEST42 is shipped at room temperature Upon receipt Storage store at 20 C Product is guaranteed for six months from date of shipment when stored properly Contents 6 ug pET DEST42 supplied at a concentration of 150 ng pl in TE pH 8 0 Accessory Products Additional Additional products that may be used with pET DEST42 are Products available from Invitrogen Ordering information is provided below Product Amount Catalog no Gateway LR Clonase Enzyme Mix 20 reactions 11791 019 One Shot TOP10 Chemically Competent Cells 10 reactions C4040 10 20 reactions C4040 03 One Shot TOP10 Electrocompetent Cells 10 reactions C4040 50 20 reactions C4040 52 Ampicillin 58 Q100 16 Carb
14. itope or a polyhistidine 6xHis tag For more information refer to our Web site www invitrogen com or contact Technical Service page 11 The C terminal peptide containing the V5 epitope and the polyhistidine tag will add approximately 4 kDa to your protein The presence of the C terminal polyhistidine 6xHis tag in your recombinant fusion protein allows use of a metal chelating resin such as ProBond to purify your fusion protein The ProBond Purification System and bulk ProBond resin are available from Invitrogen see page vi for ordering information Invitrogen also offers Ni NTA Agarose Catalog no R901 01 for purification of proteins containing a polyhistidine 6xHis tag Note Other metal chelating resins and purification methods are suitable Appendix Map and Features of pET DEST42 Map of pET The map below shows the elements of pET DEST42 DNA DEST42 from the entry clone replaces the region between bases 409 and 2092 The complete sequence of pET DEST42 is available from our Web site www invitrogen com or by contacting Technical Service page 11 gt FM cB attR2 M5 epitope 6xHis Comments for pET DEST42 7440 nucleotides T7 promoter bases 318 334 lac operator lacO bases 337 361 attR1 recombination site bases 402 526 Chloramphenicol resistance gene bases 635 1294 ccdB gene bases 1636 1941 attR2 recombination site bases 1982 2106 V5 epitope bases 2126 2167 Polyhistidine 6
15. n of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 continued on next page 13 Purchaser Notification continued Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 30 T7 Expression System Limited Use Label License No 54 ULB ccdB Selection Technology 14 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany The composition and or use of this product may be claimed in U S Patent No 5 693 489 licensed to Life Technologies Corporation by Brookhaven Science Associates LLC The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U S Department of Energy and is the subject of patents and patent applications assigned to Brookhaven Science Associates LLC BSA By provisions of the Distribution License Agreement granted to Life Technologies covering said patents and patent applications Life
16. ne should not contain a stop codon The gene should also be designed to be in frame with the C terminal epitope tag after recombination Refer to the Recombination Region on the next page e If you DO NOT wish to include the V5 epitope and 6xHis tag your gene should contain a stop codon in the entry clone Recombining Each entry clone contains attL sites flanking the gene of Your Gene of interest Genes in an entry clone are transferred to the Interest destination vector backbone by mixing the DNAs with the Gateway LR Clonase enzyme mix The resulting recombination reaction is then transformed into E coli and the expression clone selected Recombination between the attR sites on the destination vector and the attL sites on the entry clone replaces the ccdB gene and the chloramphenicol Cm gene with the gene of interest and results in the formation of attB sites in the expression clone Follow the instructions in the Gateway Technology Manual to set up the LR Clonase reaction transform a recA endA E coli strain e g TOP10 or DH5 and select for the expression clone Confirming The ccdB gene mutates at a very low frequency resulting in the a very low number of false positives True expression clones Expression will be ampicillin resistant and chloramphenicol sensitive Clone Transformants containing a plasmid with a mutated ccdB gene will be both ampicillin and chloramphenicol resistant To check your putative e
17. oosing a Selection Agent Once you have selected your E coli host strain you are ready to test for expression of your gene This section provides general guidelines for expressing and analyzing your protein of interest For detailed information on transforming your BL21 strain inducing expression with IPTG and analyzing samples refer to your specific BL21 E coli strain manual If you are using a BL21 strain from Invitrogen the manuals are available for downloading from our Web site or by contacting Technical Service page 11 The basic steps needed to induce expression of your gene in a BL21 E coli strain are outlined below 1 Isolate plasmid DNA using standard procedures and transform your construct into BL21 cells 2 Grow the transformants and induce expression with IPTG over several hours Take several time points to determine the optimal time of expression You may prepare plasmid DNA using your method of choice We recommend using the S N A P MidiPrep Kit Catalog no K1910 01 for isolation of pure plasmid DNA Note that since you are purifying a low copy number plasmid you may need to increase the amount of bacterial culture that you use to prepare your plasmid construct For most purposes ampicillin works well for selection of transformants and expression experiments However if you find that your expression level is low you may want to use carbenicillin instead The resistance gene for ampicillin
18. recombination sites other than those purchased from Life Technologies Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Life Technologies Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose The buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Life Technologies under the patents identified above to distribute such materials Transfer of such materials and or inform
19. ropagate and maintain pET DEST42 we recommend using Library Efficiency DB3 1 Competent Cells Catalog no 11782 018 from Invitrogen for transformation The DB3 1 E coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene DO NOT use general E coli cloning strains including TOP10 or DH5 for propagation and maintenance as these strains are sensitive to CcdB effects To recombine your gene of interest into pET DEST42 you should have an entry clone containing your gene of interest For your convenience Invitrogen offers the pENTR Directional TOPO Cloning Kit Catalog no K2400 20 for 5 minute cloning of your gene of interest into an entry vector For more information refer to our Web site www invitrogen com or contact Technical Service page 11 For detailed information on constructing an entry clone refer to the specific entry vector manual For detailed information on performing the LR recombination reaction refer to the Gateway Technology Manual continued on next page Using pET DEST42 continued Points to e Your insert should contain a ribosome binding site Consider AGGAG A G approximately 9 10 base pairs upstream Before of the ATG initiation codon Gold 1988 Miller 1992 This Recombining will ensure the optimal spacing for proper initiation of translation e If you wish to include the V5 epitope and 6xHis tag your gene in the entry clo
20. s The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Introduction Limited Use Label License No 19 Gateway Cloning Products pET DEST42 is covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any
21. xHis region bases 2177 2194 T7 transcription termination region bases 2209 2337 bla promoter bases 2638 2736 Ampicillin b a resistance gene ORF bases 2737 3597 pBR322 origin bases 3742 4415 ROP ORF bases 4786 4977 complementary strand lacl ORF bases 6289 7380 complementary strand continued on next page Map and Features of pET DESTA2 continued Features of pET DEST42 pET DEST42 7440 bp contains the following elements All features have been functionally tested Feature Benefit T7 promoter Allows high level IPTG inducible expression of your recombinant protein in E coli strains expressing the T7 RNA polymerase lac operator lacO Binding site for lac repressor that serves to reduce basal expression of the recombinant protein attR1 and attR2 sites Allows recombinational cloning of the gene of interest from an entry clone Chloramphenicol resistance gene Allows counterselection of expression clones ccdB gene Allows negative selection of expression clones V5 epitope Allows detection of the recombinant protein by the Anti V5 antibodies Southern et al 1991 C terminal polyhistidine tag Allows purification of the recombinant protein on metal chelating resin such as ProBond Allows detection of the recombinant protein by the Anti His C term antibodies Lindner et al 1997 T7 transcription termination region Sequence from bacteriopha
22. xpression clone test for growth on LB plates containing 30 ug ml chloramphenicol A true expression clone will not grow in the presence of chloramphenicol continued on next page Using pET DEST42 continued Recombination The recombination region of the expression clone resulting Region from pET DEST42 x entry clone is shown below Features of the Recombination Region e Shaded regions correspond to those DNA sequences transferred from the entry clone into pET DEST42 by recombination Non shaded regions are derived from the pET DESTA2 vector e The underlined nucleotides flanking the shaded region correspond to bases 409 and 2092 respectively of the pET DEST42 vector sequence T7 promoter lac operator 301 TCTCGATCCC GCGAAATTAA TACGACTCAC TATAGGGGAA TTGTGAGCGG AGAGCTAGGG CGCTTTAATT ATGCTGAGTG ATATCCCCTT AACACTCGCC 361 CCCTCTAGAA ATAATTTTGT TTAACTTTAA GAAGGAATAT CACAAGTTIG 409 GGGAGATCTT TATTAAAACA AATTGAAATT CTTCCTTATA GTGTTCAAAC 421 CAGGCTN 2 ercccan GENE ATAACAATTC TATTGTTAAG TACAAAAAAG ATOM 2092 Pro Ala Phe Leu Tyr Lys Val Val Ile NAC CCA GCT TIC TTG TAC AAA GTG GTG ATC NTG GGT CGA AAG AAC ATG TTT CAC CAC TAG l J attB2 V5 epitope attB1 Asn Ser Lys AAT TCG AAG TTA AGC TTC Leu Glu Gly Lys Pro 2120 CTT GAA GGT AAG CCT GAA CTT CCA TTC GGA Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG TAG GGA TTG GGA
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