pET-38b(+) Vector TB202 12/98

Developed through collaboration between The pET-38b(+) vector (Cat. No. 70137-3) is designed for expression of C-terminal CBDcex
Novagen and CBD Technologies, Inc. fusion proteins. Target sequences inserted between the N-terminal signal sequence and C-terminal
CBDcex•Tag™ and His•Tag® sequences allow for potential export of the expressed protein.
Sequences inserted at the Nde I site will be expressed in the cytoplasm. Note that inserts must
allow read-through in the proper reading frame (shown below) to produce the desired polypeptide
fusions. A variety of cloning sites and strategies are available. 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-38b(+) sequence landmarks

T7 promoter 818-834
Spe I(501)
T7 transcription start 817 Ahd I(502)
Signal sequence 621-746 Xho I(549)
Not I(557)
Multiple cloning sites Hind III(564)
Aat II(574)
(Nhe I - Xho I) 546-620 Pst I(580)
Pro-Thr spacer 504-524 Sse8387 I(580)
Bpu1102 I(80) BsrG I(583)
CBDcex•Tag coding seq. 180-503 Avr II(131) UbaE I(584)
Dra III(5579) Rsr II(308) EcoR I(589)
His•Tag coding sequence 150-173 BamH I(595)
Sac I(347)
T7 terminator 26-72 EcoR V(603)
Nco I(608)
810) CB
lacI coding sequence 1225-2304 355-5 Dc ( Nhe I(615)
pBR322 origin 3738 ori (5 ex 18
0-5 Nde I(745)
f1 45 Xba I(783)
Kan coding sequence 4447-5259 ) )
Pvu I(4878) SgrA I(894)
9

f1 origin 5355-5810
25

Sgf I(4878)
-5

Sph I(1050)
47

Sma I(4752)
(44
Kan

Nru I(4535)

pET-38b(+)
(5821bp)

lac
Mlu I(1575)

I
Eco57 I(4224) Bcl I(1589)

(1225-2
30 Apa I(1786)
ori

4)
(3
73

8) BssH II(1986)
Hpa I(2081)
BspLU11 I(3676)
Sap I(3560)
Bst1107 I(3447) PshA I(2420)
Tth111 I(3421)
Bgl I(2639)
Fsp I(2657)
Psp5 II(2682)

pET upstream primer #69214-3

T7 promoter lac operator Xba I rbs
ATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
CBDcexLEAD primer #70142-3
Nde I signal sequence
TATACATATGCCTCGTACTACTCCAGCTCCAGGTCATCCGGCTCGTGGTGCTCGTACTGCTCTGCGTACTACTCTGGCTGCTGCTGCTGCTACCCTGGTTGTAGGTGCAACTGTTGTTCTGCCAGCTCAGGCA
˜∏ÂÊÊ∏Å∏ÌÓ∏ÅÂÌÅÂÊÅÒÂÊÊÒÅÅÅÅÅÊÒ◊◊ÌÅÊ◊◊Ò∏ÅŒÅ
UbaE I Pst I Ahd I
signal peptidase
Nhe I Nco I EcoR V BamH I EcoR I BsrG I Sse8387 I Aat II Hind III Not I Xho I PT-spacer Spe I
GCTAGCACCATGGTGATATCGGATCCGAATTCTGTACACCTGCAGGACGTCAAGCTTGCGGCCGCACTCGAGGGTCTGGTGCCACGCGGTTCCGGTCCTCCGACGCCGACCCCGACTAGTGGTCCGGCCGGG
ÅÍʘ◊ÈÍÎ∏ˆÍ◊ÓÒŒÎ◊ÒÅÅÅÒ‰ÌÒ◊∏ÂÌÍÌ∏∏Ê∏Ê∏ÊÍÌ∏ÅÌ
thrombin
CBDcex•Tag His•Tag
TGCCAGGTGCTGTGGGGCGTCAACCAGTGGAACACC...252bp...GGCACGCCCTGCACGGTCGGCGTCGAGCACCACCACCACCACCACCACCACTAATTGATTAATA
ÇŒ◊Ò„Ì◊ˆŒ„ˆÊ... 84aa...ÌÊ∏ÇÊ◊Ì◊‰ÓÓÓÓÓÓÓÓEnd
ASCBDcex primer #70143-3
Avr II Bpu1102 I T7 terminator
CCTAGGCTGCTAAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
T7 terminator primer #69337-3

pET-38b(+) cloning/expression region

Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144

pET-38b(+) Restriction Sites TB202 12/98

Enzyme # Sites Locations Enzyme # Sites Locations Enzyme # Sites Locations

AatII 1 574 DraIII 1 5579 SgrAI 1 894
AccI 2 403 3446 DrdI 3 3369 3784 5534 SmaI 1 4752
AciI 81 DsaI 4 294 608 1012 2648 SpeI 1 501
AflIII 3 419 1575 3676 EaeI 7 317 367 491 557 883 SphI 1 1050
AhdI 1 502 1015 2249 Sse8387I 1 580
AluI 26 EagI 3 317 491 557 SspI 2 4803 5371
AlwI 13 EarI 3 1193 3560 4691 StyI 3 57 131 608
Alw26I 6 1272 1677 1803 2190 3317 Eco47III 3 980 2481 2930 TaiI 18
4894 Eco57I 1 4224 TaqI 17
AlwNI 2 482 4092 EcoNI 2 1110 4790 TfiI 9 2254 2556 2726 3230 3651
ApaI 1 1786 EcoO109I 3 53 1008 2682 4789 4845 5017 5108
ApaLI 3 1555 3490 3990 EcoRI 1 589 ThaI 39
ApoI 6 589 1850 4491 4675 5381 EcoRII 13 TseI 28
5392 EcoRV 1 603 Tsp45I 11
AvaI 2 549 4750 FauI 18 Tsp509I 21
AvaII 8 308 496 523 2127 2503 Fnu4HI 48 TspRI 13
2591 2682 2961 FokI 10 728 1621 1630 2895 2957 Tth111I 1 3421
AvrII 1 131 3035 3221 3362 4516 5122 UbaEI 1 584
BamHI 1 595 FspI 1 2657 VspI 6 139 832 2260 2319 5077
BanI 12 HaeII 14 5266
BanII 6 347 959 973 1786 4533 HaeIII 26 XbaI 1 783
5654 HgaI 15 XcmI 3 1431 1947 1965
BbsI 4 1721 2060 2434 2794 HhaI 48 XhoI 1 549
BbvI 28 HincII 2 467 2081 XmnI 2 3234 5267
BcgI 3 1867 2435 3287 HindIII 1 564
BcgI’ 3 1901 2401 3253 HinfI 18 Enzymes that do not cut pET-38b(+):
BclI 1 1589 HpaI 1 2081 AflII AscI BglII BsaI BseRI
BfaI 9 70 132 502 616 784 HphI 20 Bsu36I DraI FseI KpnI MscI
2690 4171 4478 5730 MaeIII 20 MunI NspV PacI PinAI PmeI
BglI 1 2639 MboII 13 PmlI SacII SalI SanDI ScaI
BpmI 6 743 749 1413 1902 2536 MluI 1 1575 SexAI SfiI SnaBI SrfI StuI
3203 MnlI 29 SunI SwaI
Bpu10I 3 624 2782 4895 MseI 26
Bpu1102I 1 80 MslI 7 610 1627 1915 1945 2663
BsaAI 2 3428 5579 2858 3249
BsaBI 3 848 858 2873 MspI 37
BsaHI 9 178 469 515 571 898 MspA1I 8 84 1605 2175 2268 3267
919 1033 1532 2215 3386 4018 4263
BsaJI 14 MwoI 47
BsaWI 9 2 406 525 1894 2397 NarI 4 898 919 1033 2215
2865 3882 4029 5013 NciI 14
BsgI 4 208 1426 1626 2836 NcoI 1 608
BsiEI 8 185 320 494 560 2360 NdeI 1 745
3592 4016 4878 NgoAIV 4 885 2473 2633 5680
BsiHKAI 8 175 347 705 1075 1559 NheI 1 615
2670 3494 3994 NlaIII 25
BslI 30 NlaIV 26
BsmI 2 4762 4839 NotI 1 557
BsmBI 3 2190 3317 4894 NruI 1 4535
BsmFI 4 1036 2577 2947 5794 NsiI 2 4728 4994
Bsp1286I 14 NspI 4 1050 3021 3313 3680
BspEI 2 2 2865 PflMI 2 1157 5141
BspLU11I 1 3676 PleI 9 832 1124 1211 2007 3570
BspMI 2 370 569 4055 5110 5514 5522
BsrI 22 PshAI 1 2420
BsrBI 5 412 804 3609 5277 5723 Psp1406I 4 1237 2605 3001 5364
BsrDI 2 1622 1988 Psp5II 1 2682
BsrFI 8 450 885 894 1261 2473 PstI 1 580
2633 4832 5680 PvuI 1 4878
BsrGI 1 583 PvuII 3 2175 2268 3267
BssHII 1 1986 RcaI 3 973 4396 5271
BssSI 2 707 3849 RsaI 7 585 686 698 737 1722
Bst1107I 1 3447 3482 4713
BstEII 2 357 1756 RsrII 1 308
BstXI 3 1377 1506 1629 SacI 1 347
BstYI 7 595 1139 2351 2868 4317 SapI 1 3560
4328 5127 Sau3AI 22
Cac8I 45 Sau96I 17
ClaI 2 852 4569 ScrFI 27
CviJI 97 SfaNI 23
DdeI 11 SfcI 5 576 817 3941 4132 5798
DpnI 22 SgfI 1 4878

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