Invitrogen Bac to Bac Expression System

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Map and Features of pFastBac 1, continued Features of the Vector pFastBac 1 (4775 bp) contains the following elements. All features have been functionally tested. Feature Polyhedrin promoter (P PH ) Multiple cloning site SV40 polyadenylation signal Tn7L and Tn7R f1 origin Ampicillin resistance gene pUC origin Gentamicin resistance gene Benefit Allows efficient, high-level expression of your recombinant protein in insect cells (O'Reilly et al., 1992). Allows restriction enzyme-mediated cloning of your gene of interest. Permits efficient transcription termination and polyadenylation of mRNA (Westwood et al., 1993). Mini Tn7 elements that permit site-specific transposition of the gene of interest into the baculovirus genome (i.e. bmon14272 bacmid) (Luckow et al., 1993). Allows rescue of single-stranded DNA. Allows selection of the plasmid in E. coli. Permits high-copy replication and maintenance in E. coli. Permits selection of the recombinant bacmid in DH10Bac E. coli. 54
Map and Features of pFastBac HT pFastBac HT A Map The map below shows the elements of pFastBac HT A. The complete sequences of the pFastBac HT A, B, and C vectors are available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 62). ATG 6xHis TEV Ehe II Nco I BamH I* EcoR I Stu I Sal I Sst I Spe I Not I Nsp V Xba I Pst I Xho I Sph I Kpn I Hind III Gentamicin P PH SV40 pA Tn7L Tn7R pFastBac TM HT A 4856 bp ori f1 Comments for pFastBac TM HT A 4856 nucleotides pUC ori Ampicillin f1 origin: bases 2-457 Ampicillin resistance gene: bases 589-1449 pUC origin: bases 1594-2267 Tn7R: bases 2511-2735 Gentamicin resistance gene: bases 2802-3335 (complementary strand) Polyhedrin promoter (P PH ): bases 3904-4032 Initiation ATG: bases 4050-4052 6xHis tag: bases 4062-4079 TEV recognition site: bases 4101-4121 Multiple cloning site: bases 4119-4222 SV40 polyadenylation signal: bases 4240-4480 Tn7L: bases 4509-4674 *Frameshift occurs at the BamH I site in each vector continued on next page 55
Page 1:
Bac-to-Bac ® Baculovirus Expressio
Page 5 and 6:
Kit Contents and Storage Types of P
Page 7 and 8:
Accessory Products Introduction The
Page 9 and 10:
Introduction Overview Introduction
Page 11 and 12: The Bac-to-Bac ® Baculovirus Expre
Page 13 and 14: The Bac-to-Bac ® Baculovirus Expre
Page 15 and 16: Culturing Insect Cells General Guid
Page 17 and 18: Generating the Recombinant pFastBac
Page 19 and 20: Cloning into pFastBac HT A, B, and
Page 21 and 22: Cloning into pFastBac HT A, B, and
Page 23 and 24: Cloning into pFastBac Dual Introdu
Page 25 and 26: Transformation and Analysis Introdu
Page 27 and 28: Generating the Recombinant Bacmid T
Page 29 and 30: Transforming DH10Bac E. coli, cont
Page 31 and 32: Analyzing Recombinant Bacmid DNA by
Page 33 and 34: Analyzing Recombinant Bacmid DNA by
Page 35 and 36: Transfecting Insect Cells, continue
Page 37 and 38: Isolating P1 Viral Stock Introducti
Page 39 and 40: Amplifying Your Baculoviral Stock I
Page 41 and 42: Performing a Viral Plaque Assay Int
Page 43 and 44: Performing a Viral Plaque Assay, co
Page 49 and 50: Expressing Your Recombinant Protein
Page 51 and 52: Troubleshooting Cloning into the pF
Page 53 and 54: Troubleshooting, continued Generati
Page 55 and 56: Troubleshooting, continued Isolatin
Page 57 and 58: Appendix Recipes Antibiotic Stock S
Page 59 and 60: Isolating Recombinant Bacmid DNA In
Page 61: Map and Features of pFastBac 1 pFa
Page 65 and 66: Map and Features of pFastBac Dual
Page 67 and 68: Map of pFastBac 1-Gus Description
Page 69 and 70: Map of pFastBac Dual-Gus/CAT Descr
Page 71 and 72: Technical Service, continued Limite
Page 73 and 74: Purchaser Notification, continued L
Page 75 and 76: Product Qualification, continued pF
Page 77 and 78: References Anderson, D., Harris, R.
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Invitrogen Bac to Bac Expression System

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