pET-35b(+) Vector
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pET-35b(+) Vector TB197 12/98 Developed through collaboration between Novagen and CBD Technologies, Inc. The pET-35b(+) (Cat. No. 70103-3) vector is designed for expression of CBDclos fusion proteins. A variety of cloning sites and strategies are available. Rapid directional cloning of PCR-amplified DNA is available with the pET-35 Xa/LIC Vector Kit (Cat. No. 70115-3), which contains linearized pET-35b(+) ready for annealing with appropriately prepared inserts. When cloned with the Xa/LIC method, resulting CBDclos•Tag™ fusion proteins can be cleaved precisely at the vector-encoded junction using Factor Xa. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle 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 virions 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-35b(+) sequence landmarks T7 promoter T7 transcription start CBDclos•Tag coding seq. S•Tag coding sequence Multiple cloning sites (BseR I - Xho I) His•Tag coding sequence T7 terminator lacI coding sequence pBR322 origin Kan coding sequence f1 origin 174-241 150-173 26-72 1331-2410 3844 4553-5365 5461-5916 Dra III(5685) Sma I(4858) Nru I(4641) insert clo s (38 5) 1 SgrA I(1000) pET-35b(+) lacI (1 3 3 1-2 41 0) (5927bp) Eco57 I(4330) AlwN I(4198) T4 DNA pol + dGTP o ri 84 BssS I(3955) (3 GGTATTGAGGGTCGCATG...sense..TAAGG GCGTAC...anti...ATTCCGAGATTGAGAGGAGA Xba I(889) Sph I(1156) stop (or read through) 5'GGTATTGAGGGTCGCATG...sense..TAAGGCTCTAACTCTCCTCT 3' 3'CCATAACTCCCAGCGTAC...anti...ATTCCGAGATTGAGAGGAGA 5' CB D ) 51 -8 Pvu I(4984) Sgf I(4984) Kan (45 53 -5 36 -5916) (5461 gin ori 1 f Note: Primer sequence extensions required for Xa/LIC compatibility are underlined in the diagram below. start Met (or any) Bpu1102 I(80) Avr II(131) Xho I(174) Eag I(182) Not I(182) Hind III(189) Sal I(195) Sac I(206) EcoR I(208) BamH I(214) EcoR V(222) Msc I(232) Nco I(227) BseR I(251) PinA I(273) Kpn I(280) Bgl II(283) Nsp V(310) Mun I(341) Sca I(350) Sac II(374) SexA I(378) Nde I(582) 924-940 923 381-851 291-335 4 BssH II(2092) Hpa I(2187) Sap I(3666) Bst1107 I(3553) Tth111 I(3527) CTCTAACTCTCCTCT PshA I(2526) Bgl I(2745) Fsp I(2763) Psp5 II(2788) Xa/LIC vector Xa/LIC overhangs pET upstream primer #69214-3 BstE II(1862) Apa I(1892) ) BspLU11 I(3782) + TCC AGGCCATAACTCCCA Mlu I(1681) Bcl I(1695) T7 promoter lac operator rbs Xba I ATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA CBDclos•Tag primer #70118-3 SexA I CBDclos•Tag Sac II TATACATAATGTCAGTTGAA...390bp...TCAACACCAGTTGTAAATCCAAAAGTTACAGGATATATAGGTGGAGCTAAAGTTCTTGGTACAGCACCAGGTTCCGCGGGT ˜Í◊‰...130aa...ÍÊ∏◊◊ˆ∏◊ÊÌÁÈÌÌÅ◊ÒÌÊÅ∏ÌÍÅÌ Sca I S•Tag Mun I S•Tag primer #69945-3 Nsp V LIC site Bgl II Kpn I PinA I CTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTATTGAGGGTCGC Ò◊∏ÂÌÍÊÅÈ̘‰ÊÅÅÅωŒӘÎÍ∏ÎÒÌÊÌÌÌÍÌȉÌ thrombin BseR I Msc I Nco I EcoR V BamH I EcoR I Sac I Sal I Hind III Eag I Not I Factor Xa His•Tag Xho I TCTAACTCTCCTCTGGCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAATA ͈Í∏ÒŘÅÈÍÎ∏ˆÍÍÍ◊ÎÒÅÅÅÒ‰ÓÓÓÓÓÓÓÓEnd Avr II T7 terminator Bpu1102 I CCTAGGCTGCTAAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG T7 terminator primer #69337-3 pET-35b(+) cloning/expression region Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144 pET-35b(+) Restriction Sites Enzyme AccI AciI AflIII AluI AlwI Alw26I # Sites 2 78 2 25 12 6 AlwNI ApaI ApaLI ApoI 1 1 3 10 AvaI AvaII AvrII BamHI BanI 2 5 1 1 10 BanII 6 BbsI BbvI BcgI BcgI’ BclI BfaI 4 26 4 4 1 8 BglI BglII BpmI Bpu10I Bpu1102I BsaAI BsaBI BsaHI BsaJI BsaWI 1 1 4 2 1 2 3 5 12 9 BseRI BsgI BsiEI BsiHKAI 1 3 5 7 BslI BsmI BsmBI BsmFI Bsp1286I BspEI BspLU11I BsrI BsrBI BsrDI BsrFI 26 2 3 4 12 2 1 23 4 2 8 BssHII BssSI Bst1107I BstEII BstXI BstYI 1 1 1 1 3 8 Cac8I ClaI CviJI DdeI 42 2 89 10 DpnI DraI DraIII DrdI DsaI 22 2 1 3 4 Locations 196 3552 1681 3782 1378 5000 4198 1892 1661 208 1956 174 2233 131 214 276 1601 206 5760 1827 1783 210 176 1695 70 4277 2745 283 1519 2888 80 3534 954 1004 1909 2296 3423 3596 312 4597 4856 2609 4096 573 4781 754 5487 837 5498 2697 2788 3067 361 2320 1065 1003 2450 1079 1024 2576 1892 1138 5722 4639 2166 2540 2900 1973 2007 2541 2507 3393 3359 132 4584 643 5836 890 2008 5001 2642 2796 3309 5685 964 1025 2979 1139 1638 2321 2 2971 251 1532 185 175 3600 261 3988 273 4135 2000 5119 2503 1732 2466 206 4100 2942 3698 1181 4122 1665 4984 2776 4868 2296 1142 4945 3423 2683 2 3782 2971 910 1728 273 2739 2092 3955 3553 1862 1483 214 4423 3715 2094 991 4938 958 4675 80 3590 101 4057 753 5685 3475 227 759 1612 283 4434 3890 371 5000 3053 5900 5383 5829 1000 5786 1367 1735 1245 5233 2253 4466 5640 1118 2457 2888 5001 2754 2579 2974 3050 5365 TB197 12/98 Enzyme EaeI EagI EarI Eco47III Eco57I EcoNI EcoO109I EcoRI EcoRII # Sites 5 1 3 3 1 2 3 1 10 EcoRV FauI Fnu4HI FokI 1 18 44 9 FspI HaeII HaeIII HgaI HhaI HincII HindIII HinfI HpaI HphI KpnI MaeIII MboII MluI MnlI MscI MseI MslI 1 14 24 11 46 2 1 18 1 16 1 19 13 1 27 1 30 7 MspI MspA1I 30 10 MunI MwoI NarI NciI NcoI NdeI NgoAIV NlaIII NlaIV NotI NruI NsiI NspI NspV PflMI PinAI PleI 1 39 4 12 1 1 4 27 25 1 1 2 4 1 3 1 9 PshAI Psp1406I Psp5II PvuI PvuII RcaI RsaI 1 4 1 1 3 3 7 SacI SacII SalI SapI Sau3AI Sau96I ScaI ScrFI SexAI 1 1 1 1 22 14 1 22 1 Locations 182 230 182 1299 3666 1086 2587 4330 1216 4896 53 1114 208 378 1404 3808 3929 222 1727 3327 2763 197 189 1736 3468 989 1121 2355 4797 3036 2788 1719 3942 3001 4622 2259 4872 3063 5228 2316 5229 3141 2187 2187 280 1681 232 681 2964 1733 3355 2021 2051 2769 84 2374 341 325 3373 373 3492 1711 4124 2281 4369 1004 1025 1139 2321 227 582 991 2579 2739 5786 5100 3127 3419 3786 1263 5247 1230 5216 1317 5620 2113 5628 2711 3107 5470 2374 4502 350 4819 3373 5377 389 713 182 4641 4834 1156 310 302 273 938 4161 2526 1343 2788 4984 2281 1079 278 3588 206 374 195 3666 Enzyme SfaNI SfcI SgfI SgrAI SmaI SphI SspI StyI SwaI TaiI TaqI TfiI # Sites 24 4 1 1 1 1 2 3 2 15 17 9 ThaI TseI Tsp45I 37 26 9 Tsp509I TspRI Tth111I VspI 29 13 1 6 XbaI XcmI XhoI XmnI 1 3 1 2 Locations 923 4984 1000 4858 1156 4909 57 753 4047 4238 5477 131 759 227 2360 4895 2662 4951 2832 5123 3336 5214 3757 633 3434 686 3529 1862 5131 2690 5858 3221 938 2366 2425 5183 2053 2071 3527 139 5372 889 1537 174 3340 5373 Enzymes that do not cut pET-35b(+): AatII AflII AhdI AscI BspMI BsrGI Bsu36I FseI PacI PmeI PmlI PstI SanDI SfiI SnaBI SpeI Sse8387I StuI SunI UbaEI 3676 1828 350 378 Novagen • FAX 608-238-1388 • E-MAIL [email protected] 5904 BsaI NheI RsrII SrfI
