pcDNA - Thermo Fisher Scientific
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1. 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control and 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 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 a2. GGA Leu Gly Thr Glu Leu Gly BK l F R Fock V Bek nol l 923 TCC ACT AGT CCA GTG TGG TGG AAT TCT GCA GAT ATC CAG CAC AGT GGC GGC CGC Ser Thr Ser Pro Val Trp Trp Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg SB I a Ape eae II Siy j myc epitope 977 TCG AGL CTA GAG GGC CCG CGG TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT Ser Ser Leu Glu Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Age l Polyhistidine tag Pme 7 l 1028 CTG AAT ATG CAT acc GGT CAT CAT CAC CAT CAC CAT TGA GTTT AAACCCGCTG Leu Asn Met His Thr Gly His His His His His His BGH Reverse priming site l 1 1081 ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA Continued on next page Cloning into pcDNA 3 1 myc His A B and C Continued TM Multiple Cloning Below is the multiple cloning site for pcDNA 3 1 myc His C Restriction sites are Site of Version C labeled to indicate the cleavage site The boxed nucleotides indicate the variable 861 918 969 1020 1069 region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pcDNA 3 1 myc His C is available for downloading from our website at www invitrogen com or from Technical Support see page 11 T7 promoter priming site Hind III Kpn ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TA AGC TTG GTA CCG AGC Ser Leu Val Pro Ser PRIM ES SEN ECORV TCG GAT CCA CTA GTC CAG TGT GGT GGA ATT CTG
3. His A is available for downloading from our website at www invitrogen com or from Technical Support see page 11 T7 promoter priming site Hind MI Kpn BamH i 1 861 ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TAA GCT TGG TAC CGA GCT CGG Ala Trp Tyr Arg Ala Arg Bat EGOR l EROR V BSK l iu 922 ATC CAC TAG TCC AGT GTG GTG GAA TTC TGC AGA TAT CCA GCA CAG TGG CGG CCG Ile His Ser Ser Val Val Glu Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Xho I Xba I Apa I Sfu l myc epitope l 976 CTC GAG TCT AGA GGG cg TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT Leu Glu Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Age Polyhistidine tag Fmel I 1 r 1030 ATG CAT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACCC GCTGATCAGC Met His Thr Glu His His His His His His BGH Reverse priming site Qd 1083 CTCGACTGTG CCTTCTAG TM Multiple Cloning Below is the multiple cloning site for pe DNA 3 1 myc His B Restriction sites are Site of Version B labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pcDNA 3 1 myc His B is available for downloading from our website at www invitrogen com or from Technical Support see page 11 T7 promoter priming site Hind ll Kpn l Bamm 861 ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TAAG CTT GGT ACC GAG CTC
4. Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Molec Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Evan G L Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies Specific for c myc Proto oncogene Product Mol Cell Biol 5 3610 3616 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 Transfection Nature 337 387 388 Goodwin E C and Rottman F M 1992 The 3 Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate
5. nitrogen and store at 70 C until needed If you are using ProBond resin refer to the ProBond Purification manual for details about sample preparation for chromatography If you are using another resin refer to the manufacturer s instruction for recommendations on sample preparation The C terminal peptide containing the myc epitope and polyhistidine tag will add approximately 3 kDa to the size of your protein The size of the lacZ myc His fusion protein is approximately 121 kDa Appendix pcDNA 3 1 myc His A B and C Map of The figure below summarizes the features of the pcDNA 3 1 myc His vectors pcDN A 3 1 myc The nucleotide sequence for pcDNA 3 1 myc His A is available for downloading His from www invitrogen com or from Technical Support page 11 Details of the multiple cloning sites for p DNA 3 1 myc His A B and C are shown on pages 34 Age pcDNA 3 1 myc His A B C 5 5 kb Comments for pcDNA 3 1 myc His A 5493 nucleotides There is a unique BstE Il site but no Xba lor Apa sites in version C CMV promoter bases 209 863 T7 promoter priming site bases 863 882 Multiple cloning site bases 902 999 myc epitope bases 997 1026 Polyhistidine tag bases 1042 1059 BGH reverse priming site bases 1082 1099 BGH polyadenylation signal bases 1081 1295 f1 origin of replication bases 1358 1771 SV40 promoter and origin bases 1836 2160 Neomycin resistance gene bases 2196 2990 S
6. CAG ATA TCC AGC ACA GTG Ser Asp Pro Leu Val Gln Cys Gly Gly Ile Leu Gln Ile Ser Ser Thr Val Net l xno l SIE Il xg myc epitope I GCG GCC GCT CGA GGT CAC CCA TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT Ala Ala Ala Arg Gly His Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Age Polyhistidine tag Pme FR i CTG AAT ATG CAT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACCC Leu Asn Met His Thr Gly His His His His His His BGH Reverse priming site l GCTGATCAGC CTCGACTGTG CCTTCTAGTT GC Transformation and Transfection Introduction Method of Transformation Plasmid Preparation Methods of Transfection Positive Control Detection of Fusion Proteins If you need more details about the techniques discussed refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 TOP10F DH5o and select on LB plates containing 50 100 ug ml ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert For your convenience TOP10F is available as chemically competent or electrocompetent cells from Invitrogen see page 10 We recommend that you sequence your construct to confirm that your gene is fused in frame with the myc epitope and the C terminal polyhistidine tag We suggest using the T7 Promot
7. His C term HRP Antibody BGH reverse priming site Allows sequencing through the insert Bovine growth hormone BGH polyadenylation signal Efficient transcription termination and polyadenylation of mRNA Goodwin and Rottman 1992 fl origin Allows rescue of single stranded DNA SV40 early promoter and origin Allows efficient high level expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen Neomycin Geneticin resistance gene Selection of stable transfectants in mammalian cells Southern and Berg 1982 SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pUC origin High copy number replication and growth in E coli Ampicillin resistance gene B lactamase Selection of vector in E coli pcDNA 3 1 myc His lacZ Map of Control Vector pcDNA 3 1 myc His lacZ is a 8 540 bp control vector containing the gene for B galactosidase pcDNA 3 1 myc His C was digested with EcoR V and Not I A 3 2 kb blunt Not I fragment containing the P galactosidase gene was then ligated into pcDNA 3 1 myc His C in frame with the C terminal peptide The figure below summarizes the features of the pcDNA 3 1 myc His lacZ vector The nucleotide sequence for pcDNA 3 1 myc His lacZ is available by downloading it from our website www invitrogen com or by contacting Technical S
8. Invitrogen pcDNA 3 1 myc His A B and C Catalog no V800 20 Rev Date 28 October 2010 Manual part no 28 0137 MAN0000641 ii Table of Contents Kit Contents dnd Storage teerde edet e pdt deca ahh iv INTO GU CHOI sicario H 1 OLIVO EE 1 kele CEE 2 Cloning into pcDNA 3 17 myc His A B and C teen a la 2 Transformation and Transfection nanne ensen ennvenseenernvenneenvenvennvenvensennvenserneenvenneenvencenneenvennennenn 5 ABPPENdIX dum 7 PEDNA 317 TS A D an Earn E 7 peDNA S T mye His ACL arado 9 Fess TAPER 10 Technical Support sr 11 Purchaser Notificaties iiei aa eae e EEEE Ee EE EREE EEE EE EEEE EN Ea 12 References esir erara r e cia 13 iii Kit Contents and Storage Shipping and pcDNA 3 1 myc His vectors are shipped on wet ice Upon receipt store vectors at Storage 20 C Kit Contents All vectors are supplied in aliquot detailed below Store the vectors at 20 C Vector Composition Amount pcDNA 3 1 myc His A B and C 40 ul of 0 5 ug pl vector in 10 mM Tris HCl 20 ug 1 mM EDTA pH 8 0 pcDNA 3 1 myc His lacZ 40 ul of 0 5 ug ul vector in 10 mM Tris HCl 20 ug 1 mM EDTA pH 8 0 Overview Description of the System Experimental Outline Introduction pcDNA 3 1 myc His A B and C are 5 5 kb vectors derived from pcDNA 3 1 and designed for high level expression purification and detection
9. Polyadenylation J Biol Chem 267 16330 16334 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Southern P J and Berg P 1982 Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under Control of the SV40 Early Region Promoter J Molec Appl Gen 1 327 339 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2009 2010 Life Technologies Corporat
10. V40 polyadenylation signal bases 3166 3296 pUC origin bases 3679 4352 Ampicillin resistance gene bases 4497 5357 complementary strand There is a unique Sac Il site between the Apa site and the Sfu I site in version B only pcDNA 3 1 myc His A B and C Continued Features of pcDNA 3 1 myc His pcDNA 3 1 myc His A 5 493 bp pcDNA 3 1 myc His B 5 497 bp and pcDNA 3 1 myc His C 5 489 bp contain the following elements All features have been functionally tested Feature Benefit Human cytomegalovirus CMV immediate early promoter enhancer Allows efficient high level expression of your recombinant protein Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 17 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert Multiple cloning site in three reading frames Allows insertion of your gene and facilitates cloning in frame with the myc epitope and polyhistidine C terminal tag myc epitope c myc Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Allows detection of your recombinant protein with the Anti myc Antibody or Anti myc HRP Antibody Evan et al 1985 C terminal polyhistidine tag Allows purification of your recombinant protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody and the Anti
11. ccordance with instructions 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 please 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 11 Purchaser Notification Limited Use Label This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign License No 22 equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Vectors and Ltd Basel Switzerland and is provided only for use in research Information about Clones Encoding licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 Histidine Hexamer D 40724 Hilden Germany 12 References Andersson S Davis D L Dahlb ck H J rnvall H and
12. en For more information on Lipofectamine 2000 and other transfection reagents available visit our website at www invitrogen com or contact Technical Support page 11 pcDNA 3 1 myc His lacZ is provided as a positive control vector for mammalian transfection and expression see page 9 It may be used to optimize transfection conditions for your cell line The gene encoding galactosidase is expressed in mammalian cells under the CMV promoter A successful transfection will result in B galactosidase expression that can be easily assayed see below A number of antibodies are available from Invitrogen see page 10 that can be used to detect expression of your fusion protein from pcDNA 3 1 myc His Continued on next page Transformation and Transfection Continued Assay for p galactosidase Activity Geneticin Selection Guidelines Preparing Cells for Lysis Lysis of Cells Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 TOP10F DH5o and select on LB plates containing 50 100 ug ml ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert Geneticin is available separately from Invitrogen Geneticin blocks protein synthesis in mammalian cells by interfering with ribosomal function It is an aminoglycoside similar in structure to neomycin gentamycin and kanamycin Expression of the bacterial aminoglycoside phosphotransfera
13. er and BGH Reverse primer sequences Refer to the diagrams on pages 3 4 for the sequence and location of the primer binding sites For your convenience Invitrogen offers a custom primer synthesis service For more information visit www invitrogen com or contact Technical Support page 11 Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Miniprep Kit or TM the PureLink HiPure Midiprep Kit see page 10 for ordering information For established cell lines e 2 HeLa consult original references or the supplier of your cell line for the optimal method of transfection It is recommended that you follow exactly the protocol for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Methods of transfection include calcium phosphate Chen amp Okayama 1987 Wigler et al 1977 lipid mediated Felgner et al 1989 Felgner amp Ringold 1989 and electroporation Chu et al 1987 Shigekawa amp Dower 1988 For high efficiency transfection in a broad range of mammalian cells we recommend using Lipofectamine 2000 Reagent available from Invitrog
14. ion All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 13 Notes 14 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech supportainvitrogen com For country specific contact information visit our web site at www invitrogen com
15. nt cells from Invitrogen see page 10 TM To propagate and maintain the pcDNA 3 1 myc His vectors use the supplied 0 5 ug ul stock solution in TE pH 8 0 to transform a recA end A E coli strain like TOP10 TOP10F DH5a JM109 or equivalent Select transformants on LB plates containing 50 100 ug ml ampicillin Be sure to prepare a glycerol stock of each plasmid for long term storage Your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1990 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while having neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG Continued on next page Cloning into pcDNA 3 1 myc His A B and C Continued TM Multiple Cloning Below is the multiple cloning site for pcDNA 3 1 myc His A Restriction sites Site of Version A are labeled to indicate the cleavage site Note that there is a stop codon between the BamH I site and the BstX I site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of p DNA 3 1 myc
16. of recombinant proteins in mammalian hosts High level stable and non replicative transient expression can be carried out in most mammalian cells The vectors contain the following elements e Three reading frames to facilitate in frame cloning with a C terminal tag encoding the myc c myc epitope and a polyhistidine metal binding peptide e Human cytomegalovirus immediate early CMV promoter for high level expression in a wide range of mammalian cells e Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen e g COS7 The control plasmid pcDNA 3 1 myc His lacZ is the p DNA 3 1 myc His C vector with a 3 2 kb fragment containing the B galactosidase gene cloned in frame with the C terminal peptide see page 9 It is included for use as a positive control for transfection expression purification and detection in the cell line of choice Use the following outline to clone and express your gene of interest in pcDNA 3 1 myc His e Consult the multiple cloning sites described on pages 3 4 to determine which vector A B or C should be used to clone your gene in frame with the C terminal myc epitope and the polyhistidine tag e Ligate your insert into the appropriate vector and transform into E coli Select transformants on 50 100 pg ml ampicillin e Analyze your transformants for the presence of insert by restriction digestion e Select a transformant with the correc
17. of your fusion protein from pcDNA 3 1 myc His The table below describes the antibodies available and ordering information The amount supplied is sufficient for 25 Westerns Product Purpose Catalog no Anti myc Detects 10 amino acid epitope derived R950 25 from c myc Anti myc HRP See above Provided as an HRP R951 25 conjugate for time saving detection Anti His C term Detects the C terminal polyhistidine R930 25 tag requires the free carboxyl group for detection Anti His C term HRP See above Provided as an HRP R931 25 conjugate for time saving detection Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support 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 website 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
18. se gene APH derived from Tn5 in mammalian cells results in detoxification of Geneticin Southern and Berg 1982 Use as follows e Prepare Geneticin in a buffered solution e g 100 mM HEPES pH 7 3 e Use 100 1000 ug ml of Geneticin in complete medium e Calculate concentration based on the amount of active drug check lot label e Test varying concentrations of Geneticin on your cell line to determine the concentration that kills your cells kill curve Cells differ in their susceptibility to Geneticin Cells will divide once or twice in the presence of lethal doses of Geneticin so the effects of the drug take several days to become apparent Complete selection can take from 3 to 6 weeks of growth in selective medium Use the procedure below to prepare cells for lysis prior to purification of your protein on ProBond You will need 5 x 10 to 1 x 107 cells for purification of your protein on a 2 ml ProBond column see the ProBond Purification manual 1 Seed cells in five T 75 flasks or two to three T 175 flasks 2 Grow the cells in selective medium until they are 80 9076 confluent 3 Harvest the cells by treating with trypsin EDTA for 2 5 minutes or by scraping the cells in PBS 4 nactivate the trypsin by diluting with fresh medium if necessary and transfer the cells to a sterile microcentrifuge tube 5 Centrifuge the cells at 1 500 rpm for 5 minutes You may lyse the cells immediately or freeze in liquid
19. t restriction pattern and confirm that your gene is in frame with the C terminal peptide by sequencing e Transfect your construct into the cell line of choice using your own method of transfection e Test for expression of your recombinant gene by western blot analysis or functional assay If you do not have an antibody to your protein Invitrogen offers the Anti myc antibody or the Anti His C term antibody to detect your recombinant protein see page 10 e To purify your recombinant protein you may use a metal chelating resin TM TM such as ProBond ProBond resin is available separately see page 10 Methods Cloning into pcDNA 3 1 myc His A B and C General Molecular Biology Techniques E coli Strain Maintenance of pcDNA 3 1 myc His Important For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 TM Many E coli strains are suitable for the propagation of pcDNA 3 1 myc His vectors including TOP10 TOP10F DH5 T1 We recommend that you propagate vectors containing inserts in E coli strains that are recombinant deficient recA and endonuclease A deficient end A For your convenience TOP10F is available as chemically competent or electrocompete
20. upport page 11 myc epitope Age pcDNA 3 1 myc His lacZ Comments for pcDNA 3 1 myc His lacZ 8540 nucleotides CMV promoter bases 209 863 T7 promoter priming site bases 863 882 lacZ with C terminal tag 963 4106 lacZ ORF bases 963 4019 myc epitope bases 4044 4073 Polyhistidine tag bases 4089 4106 BGH reverse priming site bases 4129 4146 BGH polyadenylation signal bases 4128 4342 f1 origin of replication bases 4405 4818 SV40 promoter and origin bases 4883 5207 Neomycin resistance gene bases 5243 6037 SV40 polyadenylation signal bases 6213 63473 pUC origin bases 67267 7399 Ampicillin resistance gene bases 7544 8404 complementary strand Accessory Products Introduction Detection of Fusion Proteins 10 The following additional products may be used with the pcDNA 3 1 myc His vectors For more information www invitrogen com or contact Technical Support see page 11 Product Amount Catalog no ProBond Purification System 6 purifications K850 01 i 50 ml R801 01 ProBond Resin 150 ml R801 15 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 Electrocomp TOP10F 2 x 20 rxns C665 11 6 x 20 rxns C665 24 One Shot TOP10F chemically competent cells 21 x 50 pl C3030 03 A number of antibodies are available from Invitrogen that can be used to detect expression
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