pET-28a-c( ) Vectors

pET-28a DNApET-28b DNApET-28c DNA

Cat. No.698-369865-369866-3

TB074 12/98

The pET-28a-c(+) vectors carry an N-terminal His•Tag®/thrombin/T7•Tag®configuration plusan optional C-terminal His•Tag sequence. Unique sites are shown on the circle map. Note that thesequence is numbered by the pBR322 convention, so the T7 expression region is reversed on thecircular map. The cloning/expression region of the coding strand transcribed by T7 RNA poly-merase is shown below. The f1 origin is oriented so that infection with helper phage will producevirions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).

pET-28a(+) sequence landmarks

Pvu I(4426)Sgf I(4426)Sma I(4300)Kan (3995-4807)T7 promoter

T7 transcription startHis•Tag coding sequenceT7•Tag coding sequenceMultiple cloning sites(BamH I - XhoI)

His•Tag coding sequenceT7 terminator

lacIcoding sequencepBR322 origin

Kan coding sequencef1 origin

370-386369270-287207-239

Dra III(5127)Bpu1102 I(80)Xho I(158)Not I(166)Eag I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)Nhe I(231)Nde I(238)Nco I(296)Xba I(335)Bgl II(401)SgrA I(442)Sph I(598)158-203140-15726-72773-18523286

3995-48074903-5358

03-5358) (49nigori 1fThe maps for pET-28b(+) and pET-28c(+)

are the same as pET-28a(+) (shown) withthe following exceptions: pET-28b(+) is a5368bp plasmid; subtract 1bp from each sitebeyond BamH I at 198. pET-28c(+) is a

5367bp plasmid; subtract 2bp from each sitebeyond BamH I at 198.

Mlu I(1123)Bcl I(1137)BstE II(1304)Apa I(1334)BssH II(1534)EcoR V(1573)Hpa I(1629)73-1852)lacI (7Cla I(4117)Nru I(4083)pET-28a(+)(5369bp)Eco57 I(3772)AlwN I(30) (3orBssS I(3397)BspLU11 I(3224)Sap I(3108)Bst1107 I(2995)Tth111 I(2969)i286)PshA I(1968)Bgl I(2187)Fsp I(2205)Psp5 II(2230)T7 promoter primer #69348-3pET upstream primer #69214-3T7 promoterlac operatorrbsBgl IIXba IAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGANde INhe IT7•TagHis•TagNco ITATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAA MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisMetAlaSerMetThrGlyGlyGlnGlnthrombinEag IHis•TagBamH IEcoR ISac INot IXho ISal IHind IIIATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28a(+)MetGlyArgGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd...GGTCGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28b(+)...GlyArgAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd...GGTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28c(+)...GlyArgIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThrLysPro...T7 terminatorBpu1102 IGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGT7 terminator primer #69337-3pET-28a-c(+) cloning/expression regionNovagen •ORDERING800-526-7319 •

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pET-28a(+) Restriction Sites

EnzymeAccIAceIII# SitesLocations21802994701618317967AciI77AflIII211233224AluI22AlwI13Alw21I715919030423542Alw44I311033038AlwNI130ApaI11334ApaBI1807ApoI619213984940AvaI21584298AvaII516752051BamHI1198BanI9253445176212BanII61905075202BbsI412691608BbvI27BccI14Bce83I62119373854BcefI629835153BcgI916019419491983BclI11137BfaI77023240265278BglI12187BglII1401BmgI11332BpmI49611450Bpu10I223304443Bpu1102I180BsaAI229765127BsaBI3400406BsaHI54467BsaJI105729621963384BsaWI72144235774561BsaXI217825075BsbI229405034BscGI11BsgI39741174BsiI13397BsiEI51691908BslI23BsmI243104387BsmAI682012254442BsmBI317382865BsmFI45842125BsoFI48Bsp24I12Bsp1286I12BspEI222413BspGI12750BspLU11I13224BsrI22BsrBI43563157BsrDI211701536BsrFI743344243805228BssHII11534Bst1107I12995194927332874EnzymeBstEIIBstXIBstYICac8ICjeICjePIClaICviJICviRIDdeIDpnIDraIIIDrdIDrdIIDsaIEaeIEagIEarIEciIEco47IIIEco57IEcoNIEcoO109IEcoRIEcoRIIEcoRVFauIFokIFspIGdiIIHaeIHaeIIHaeIIIHgaIHgiEIIHhaIHin4IHincIIHindIIIHinfIHpaIHphIMaeIIMaeIIIMboIIMluIMmeIMnlIMseIMslIMspIMspA1I5342MwoINarINciINcoINdeINgoAIVNheINlaIIINlaIVNotINruINsiINspI39412114126221124# Sites1394026301862211211323413331231101179146142411247321181161416121725256299Locations130492510541321982416386511774013876687467519EnzymePfl1108IPflMIPleIPshAIPsp5IIPsp1406IPvuIPvuIIRcaIRsaISacISalISapISau96ISau3AIScrFISfaNISfcISgfISgrAISmaISphISspIStyITaqITaqIITfiI1169276922051668514513117829104312172244340563323925054670179732502583ThaITseITsp45I3702Tsp509ITth111ITth111II3810411216294654UbaJIVspIXbaIXcmIXhoIXmnI2018215131238277# Sites12911413331111421222341111221569Locations201070546384672360346581968223078521534426172318165213944127030301901793108759506215555070TB074 12/98

31182549281548194261491262335381107221841174039422349292139466201852119822230580511334234225091043408121071610228280133633153726141528352238361347451449512729178462961661667419005283772658531922563250157333325132560431310832982029433855684633715082219656342393444247817972230116133841701431417584671369442424300598435157103150311802433734368053491929612492104439319222274456531262778465680319937191304457329699623853384335979158278221325300266328762971208427511655426218081495481526854318671513381438217211022181173162946254814242158156042971945108056298241317631758469934302384314035442611233439463336235104406842624624Enzymes that do not cut pET28a(+):AatIIAflIIAgeIBaeIBsaIBseRIBsu36IDraIEam1105IMscIMunINspVPmlIPstIRleAIScaISexAISfiISrfISse8387IStuIAscIBspMIFseIPacIRsrIISnaBISunIAvrIIBsrGIKpnIPmeISacIISpeISwaI135144422495173828651175279784281544629623843323114631493221124062293446711533566581172338111763181620212181522848258095271202121811608342765984542256928613228Novagen •FAX608-238-1388 •E-MAILnovatech@novagen.com