pBAD/Myc - Gene Synthesis

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pBAD/His Vectors, continuedMap of pBAD/HisThe figure below summarizes the features of the pBAD/His vector. Complete sequencesfor all three pBAD/His vectors are available for downloading from our World Wide Website (www.invitrogen.com) or by contacting Technical Service (see page 27). Details ofeach multiple cloning site are shown on pages 9-11.Xho ISac I*Bgl IIPst IPvu II*Kpn IEcoR ISfu IHind IIIPBADATG6xHis Xpress EKEpitope SiteMCS termaraCpBAD/HisA,B,C4.1 kbAmpicillinComments for pBAD/His A4102 nucleotidespBR322 oriaraBAD promoter region: bases 4-276Initiation ATG: bases 319-321Polyhistidine tag: bases 331-348Xpress epitope: bases 388-411Enterokinase recognition site: bases 397-411Multiple cloning site: bases 430-470rrnB transcription termination region: bases 553-710Ampicillin ORF: bases 989-1849pBR322 origin: bases 1994-2667AraC ORF: bases 4076-3198* Sac I and Pvu II are notpresent in version C.4
pBAD/Myc-His VectorFeatures ofpBAD/Myc-HisThe important elements of pBAD/Myc-His A (4094 bp), pBAD/Myc-His B (4092 bp),and pBAD/Myc-His C (4093 bp) are described in the following table. All features havebeen functionally tested.FeaturearaBAD promoter (P BAD)Optimized ribosome binding siteInitiation ATGMultiple cloning siteC-terminal myc epitope tag(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)C-terminal polyhistidine regionrrnB transcription termination regionBenefitProvides tight, dose-dependent regulation ofheterologous gene expression (Guzman etal., 1995)Increases efficiency of recombinant fusionprotein expressionProvides a translational initiation site for thefusion proteinAllows insertion of your gene for expressionAllows detection of the fusion protein by theAnti-Myc Antibody (Catalog no. R950-25)(Evans et al., 1985)Forms metal-binding site for affinitypurification of recombinant fusion proteinon metal-chelating resin (i.e. ProBond )In addition, it allows detection of therecombinant protein with Anti-His (C-term)Antibody (see page 2)Strong transcription termination regionAmpicillin resistance gene (β-lactamase) Allows selection of the plasmid in E. colipBR322 originaraC geneLow copy replication and growth in E. coliEncodes the regulatory protein for tightregulation of the P BAD promoter (Lee, 1980;Schleif, 1992)continued on next page5
Page 3 and 4: Table of ContentsTable of Contents.
Page 5 and 6: IntroductionOverviewIntroductionThe
Page 7: pBAD/His VectorFeatures ofpBAD/HisT
Page 11 and 12: MethodsGeneral CloningIntroductionT
Page 13 and 14: Cloning into pBAD/His, continuedpBA
Page 15 and 16: Cloning into pBAD/His, continuedpBA
Page 17 and 18: Cloning into pBAD/Myc-His, continue
Page 19 and 20: Cloning into pBAD/Myc-His, continue
Page 21 and 22: ExpressionIntroductionSince each re
Page 23 and 24: Expression, continuedPreparation of
Page 25 and 26: PurificationScale-up ofExpression f
Page 27 and 28: pBAD/Myc-His/lacZDescriptionpBAD/My
Page 29 and 30: Recipes, continuedSOB Medium(with A
Page 32: \ha\jZsZ_lky b kh[bjZ_l aZdZau gZ l

pBAD/Myc - Gene Synthesis

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