pET28a-c(+)質(zhì)粒圖譜

1、TB074 12/98pET-28a-c(+) VectorsCat. No.pET-28a DNA69864-3pET-28b DNA69865-3pET-28c DNA69866-3Call 137-649-32037 to buy the vector in China.The pET-28a-c(+) vectors carry an N-terminal His?Tag ?/thrombin/T7?Tag - con?guration plus an optional C-terminal His?Tag sequence. Unique sites are shown on the

2、 circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage wil

3、l produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, singlestranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).pET-28a(+) sequence landmarksT7 promoter370-386T7 transcription start369His?Tag coding sequence270-287T7?

4、Tag coding sequence207-239Multiple cloning sites(Bam H I - Xho I)158-203His?Tag coding sequence140-157T7 terminator26-72lacI coding sequence773-1852pBR322 origin3286Kan coding sequence3995-4807f1 origin4903-5358The maps for pET-28b(+) and pET-28c(+) are the same as pET-28a(+) (shown) with the follow

5、ing exceptions: pET-28b(+) is a 5368bp plasmid; subtract 1bp from each site beyond Bam H I at 198. pET-28c(+) is a 5367bp plasmid; subtract 2bp from each site beyond Bam H I at 198.Bpu1102 I (80)Dra 111(5127)- (4903-5358) f1org(Xho I (158) Not I (166) Eag I (166)Hind III (173) Sal I(179) Sac I (190)

6、 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)Pvu I (4426)Sgf I (4426)Sma I (4300)iCla I(4117)Nru I(4083)Eco57 I (3772)AlwN I (3640)BssS I (3397)BspLU11，）oriI (3224)Sap I (3108)Bst1107 I (2995)Tth111 I (2969)pET32a+)T7 promoter pri

7、mer #69348-3 pET upstream primer #69214-3 Bgl IIT7 promoterlac operatorXba IlMlu I (1123)Bcl I(1137) 7訕BstE II (1304) Apa I(1334)F BssH II (1534)EcoR V (1573)Hpa I (1629)PshA I (1968)< - Bgl I(2187) Fsp I (2205)Psp5 II(2230)rbsNco I BamH I EcoR I Sac I Sal I Hind III Not I Xho IATGGGTCGCGGATCCGAA

8、TTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28a(+) MetGlyArgGlySerGluPheGluLeuArgArgGl nAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysE nd.GlyArgAspProAs nSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisE ndAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGG

9、AATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAHis?TagNde I Nhe IT7?TagTATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAMetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisMetAlaSerMetThrGlyGlyGl nGlnEag1 thrombinSal I Hind III Not I X

10、ho IHis?Tag.GGTCGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-；8b(+).GGTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28c(+) GlyArglleArglleArgAlaPrcSerThrSerleuArgPrcHisSerSerThrThrThrThrThrThrGlulleAr

11、gleuleuThrlysPrcBpu 1102 IT7 termi natorGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGT7 termi nator primer #69337-3pET-28a-c(+) clonin g/expressi on regi onNovagen ? orderinqboo-526-7319 ? technicaisupport800-207-0144TB074 12/98pET-28a(+) Restriction Sites

12、En zyme# SitesLocati onsEn zyme# SitesLocati onsEn zyme# SitesLocati onsAccI21802994BstEll11304P?1108l12010Acelll7890161819492733287,BstXl392510541177P?MI2705468931764967BstYl91321984016871899Plel938467275915553118Acil7724163865387646753603465850625070A?III211233224Cac8l40PshAI11968AluI22Cjel26Psp5I

13、I12230Alwl13CjePI30Psp1406l4785215325494912Alw21l71591906231107221Clal14117Pvul1442630423542CviJI86Pvull3172318162815Alw44l3110330383538CviRI22Rcal352139444819AlwNI13640Ddel11Rsal3127030304261Apal11334Dpnl21Sacl1190ApaBI1807139840394223Dralll15127Sall1179Apol6192492$Drdl3291733325082Sapl131084940Drd

14、ll28465132Sau96l14Aval21584298Dsal32965602196Sau3AI21Avall516752051213922302509Eael41664315631797ScrFI22BamHl1198Eagl1166SfaNI23Ba nl92534454665801043Earl374131084239Sfcl43693489368053461762189220185164Ecil390032983444Sgfl14426Banll619050752113344081Eco47lll352820292478SgrAI14425202Eco57l13772Smal14

15、300Bbsl41269160819822342EcoNI26584338Sphl1598Bbvl27EcoO109I3535562230Sspl243514919Bccl14EcoRI1192Styl257296Bce83l6211937210733153613EcoRII10256846116117011758Taql15385432503371338443144671Taqll610311249192231264680Bcefl6642983161037264745EcoRV1157350315153Faul17T?l918022104227427783199Bcgl9160194228

16、14151449Fokl911691178244325052583433743934565465619491983280128352769291040644670Thal38Bcll11137Fspl12205Tsel27Bfal77023233622383719Gdill41664315631797Tsp45l71304213226632876297140265278Hael6851217232393250370245735300Bgll121874513Tsp509l20Bglll1401Haell14Tth111l12969Bmgl11332Haelll24Tth111ll8962165

17、5268538143821Bpml4961145020842751Hgal11385342624389Bpu10l223304443HgiEII27213810UbaJI21Bpu1102l180Hhal47Vspl53841808186746254814BsaAl229765127Hi n4l3102241124654Xbal1335BsaBl34004062421Hi ncll21811629Xcml397914951513BsaHl544646758110801763Hi ndHI1173Xhol1158BsaJl10572965605661758Hi nfl18Xmnl22782481

18、521963384429742984699Hpal11629BsaWl721442194524133430Hphl16En zymes that do not cut pET28a(+):35774561Maell14AatllA?llAgelAsclAvrllBsaXl217825075Maelll16BaelBsalBseRIBspMIBsrGIBsbl229405034Mboll12Bsu36lDralEam1105IFseIKp nlBscGl11Mlul11123MsclMu nlNspVPaclPmelBsgl397411742384Mmel734393623406842624624PmllPstlRleAlRsrllSacllBs