pET28a-c(+)PKPROMOTER质粒图谱

1、TB074 12/98pET-28a DNApET-28b DNApET-28c DNACat. No. 69864-3 69865-3 69866-3Call 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 circle map. Not

2、e 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 will produce virion

3、s 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(+) seque nce Ian dmarks T7 promoterT7 transcription start His?Tag coding sequence T7?Tag coding sequence Multiple

4、 cloning sites (Bam H I - Xho I) His?Tag coding sequence T7 terminator lacI coding sequence pBR322 origin Kan coding sequence f1 origin370-386369270-287207-239158-203140-15726-72773-185232863995-48074903-5358The maps for pET-28b(+) and pET-28c(+) are the same as pET-28a(+) (shown) with the following

5、 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.Dra III (5127)Sma I (4300)KPvu I (4426)Sgf I (4426)903-5358)f tBpu1102 I (80)Xba I (335)Bgl II (401) y _ SgrA I (442)Sph I

6、 (598)Xho I (158) Not I (166) Eag I (166)Hind Ill (173) Sal 1(179) Sac I (190)EcoR I (192) BamH I (198)Nhe I (231)Nde I (238)Nco I (296)Cla 1(4117)Nru 1(4083)TpeT32B+)T7 promoter primer #69348-3 pET upstream primer #69214-3 Bgl IIT7 promoterIEco57 I (3772)AlwN I (3640)oriBssS I (3397)BspLU11I (3224)

7、Sap 1(3108)Bst1107 I (2995)Tth111 I (2969)lac operatorXba IBstE II (1304) Apa I(1334)PshA I (1968)Mlu I (1123)Bgl 1(2187)Fsp I (2205)Psp5 11(2230)BssH II (1534)J卜 EcoR V (1573)Hpa I (1629)rbspET-28a-c(+) VectorsAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACT

8、TTAAGAAGGAGANco IHis?TagNde I Nhe IT7?TagTATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAMetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisMetAlaSerMetThrGlyGlyGl nGlnEag1 thrombin BamH I EcoR I Sac I Sal I Hind III Not I Xho IATGGGTCGCGGATCCGAA

9、TTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28a(+) MetGlyArgGlySerGluPheGluLeuArgArgGl nAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysE ndSal I Hind III Not I Xho IHis?Tag.GlyArgAspProAs nSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisE nd.GGTCGGG

10、ATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-；8b(+).GGTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28c(+) GlyArglleArglleArgAlaPrcSerThrSerleuArgPrcHisSerSerThrThrThrThrThrThrGlulleArgleuleuThrlysPrcBpu

11、 1102 IT7 terminatorGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGT7 termi nator primer #69337-3pET-28a-c(+) clonin g/expressi on regi onNovagen ? orderinqboo-526-7319 ? technicaisupport800-207-0144TB074 12/98pET-28a(+) Restriction SitesEn zyme# SitesLocati

12、 onsEn zyme# SitesLocati onsEn zyme# SitesLocati onsAccI21802994BstEll11304P?1108l12010Acelll7890161819492733287,BstXl392510541177P?MI2705468931764967BstYl91321984016871899Plel938467275915553118Acil7724163865387646753603465850625070A?III211233224Cac8l40PshAI11968AluI22Cjel26Psp5II12230Alwl13CjePI30P

13、sp1406l4785215325494912Alw21l71591906231107221Clal14117Pvul1442630423542CviJI86Pvull3172318162815Alw44l3110330383538CviRI22Rcal352139444819AlwNI13640Ddel11Rsal3127030304261Apal11334Dpnl21Sacl1190ApaBI1807139840394223Dralll15127Sall1179Apol6192492$Drdl3291733325082Sapl131084940Drdll28465132Sau96l14Av

14、al21584298Dsal32965602196Sau3AI21Avall516752051213922302509Eael41664315631797ScrFI22BamHl1198Eagl1166SfaNI23Ba nl92534454665801043Earl374131084239Sfcl43693489368053461762189220185164Ecil390032983444Sgfl14426Banll619050752113344081Eco47lll352820292478SgrAI14425202Eco57l13772Smal14300Bbsl4126916081982

15、2342EcoNI26584338Sphl1598Bbvl27EcoO109I3535562230Sspl243514919Bccl14EcoRI1192Styl257296Bce83l6211937210733153613EcoRII10256846116117011758Taql15385432503371338443144671Taqll610311249192231264680Bcefl6642983161037264745EcoRV1157350315153Faul17T?l918022104227427783199Bcgl916019422814151449Fokl91169117

16、8244325052583433743934565465619491983280128352769291040644670Thal38Bcll11137Fspl12205Tsel27Bfal77023233622383719Gdill41664315631797Tsp45l71304213226632876297140265278Hael6851217232393250370245735300Bgll121874513Tsp509l20Bglll1401Haell14Tth111l12969Bmgl11332Haelll24Tth111ll89621655268538143821Bpml496

17、1145020842751Hgal11385342624389Bpu10l223304443HgiEII27213810UbaJI21Bpu1102l180Hhal47Vspl53841808186746254814BsaAl229765127Hi n4l3102241124654Xbal1335BsaBl34004062421Hi ncll21811629Xcml397914951513BsaHl544646758110801763Hi ndHI1173Xhol1158BsaJl10572965605661758Hi nfl18Xmnl2278248152196338442974298469

18、9Hpal11629BsaWl721442194524133430Hphl16En zymes that do not cut pET28a(+):35774561Maell14AatllA?llAgelAsclAvrllBsaXl217825075Maelll16BaelBsalBseRIBspMIBsrGIBsbl229405034Mboll12Bsu36lDralEam1105IFseIKp nlBscGl11Mlul11123MsclMu nlNspVPaclPmelBsgl397411742384Mmel734393623406842624624PmllPstlRleAlRsrllSacllBsil