pET-20b(+) Vector

TB037 12/98
The pET-20b(+) vector (Cat. No. 69739-3) carries an N-terminal pelB signal sequence for
potential periplasmic localization, plus optional C-terminal HisTag sequence. 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 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 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).
Xho I(158)
Not I(166)
Eag I(166)
Hind III(173)
Sal I(179)
Hinc II(181)
Sac I(190)
EcoR I(192)
BamH I(198)
EcoR V(217)
Nco I(220)
BseR I(260)
BspM I(268)
Nde I(288)
Xba I(326)

Bpu1102 I(80)
pET-20b(+) sequence landmarks

T7 promoter
T7 transcription start
pelB coding sequence
Multiple cloning sites
(Nco I - Xho I)
HisTag coding sequence
T7 terminator
pBR322 origin
bla coding sequence
f1 origin

353-369
352
224-289
Drd II(3479)
Dra III(3474)
BsaX I(3422)

158-225
140-157
26-72
1500
2261-3118
3250-3705

f1

5)
0-370
325
(
n
igi
or

Bgl II(384)
Bpu10 I(544)
Bbs I(556)
Bsg I(598)
Eco47 III(692)

Sca I(2811)
Pvu I(2701)

Pst I(2576)

-3118)
(2261
Ap

BsaH I(2868)

pET-20b(+)

BspG I(964)

(3716bp)

Pvu II(1029)
BsmB I(1079)
Tth111 I(1183)
Bst1107 I(1209)

Pfl1108 I(2349)
Eam1105 I(2331)

HgiE II(2024)

15
ri (

00

Sap I(1322)
BspLU11 I(1438)
Afl III(1438)

AlwN I(1854)

T7 promoter primer #69348-3

T7 promoter

Bgl II

rbs

Xba I

AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
Nde I

BspM I

pelB leader

Nco I EcoR V

BamH I EcoR I Sac I

TATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGATATCGGAATTAATTCGGATCCGAATTCGAGCTCC
MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAlaMetAspIleGlyIleAsnSerAspProAsnSerSerSer
Hinc II
Sal I Hind III

Eag I
Not I

Xho I

HisTag

signal peptidase

Bpu1102 I

GTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAAC
ValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd
T7 terminator primer #69337-3

T7 terminator

TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG

pET-20b(+) cloning/expression region

Novagen ORDERING 800-526-7319

TECHNICAL SUPPORT

800-207-0144

pET-20b(+) Restriction Sites

Enzyme
AccI
AceIII
AciI
AflIII
AluI
AlwI
Alw21I

# Sites
2
5
49
1
18
13
7

Alw44I
AlwNI
ApoI
AvaI
AvaII
BamHI
BanI
BanII
BbsI
BbvI
BccI

3
1
3
2
5
1
2
2
1
26
6

Bce83I
BcefI
BcgI

5
2
6

BfaI

BglI
BglII
BpmI
Bpu10I
Bpu1102I
BsaI
BsaAI
BsaBI
BsaHI
BsaJI
BsaWI
BsaXI
BsbI
BscGI

2
1
2
1
1
2
2
2
1
4
5
1
2
10

BseRI
BsgI
BsiI
BsiEI

1
1
2
6

BslI
BsmAI
BsmBI
BsmFI
BsoFI
Bsp24I
Bsp1286I

12
4
1
2
38
4
8

BspEI
BspGI
BspLU11I
BspMI
BsrI
BsrBI
BsrDI
BsrFI
Bst1107I
BstYI

2
1
1
1
14
3
2
3
1
10

Cac8I
CjeI

16
10

CjePI

10

CviJI
CviRI

57
13

Locations
180
1208
947
1088

Enzyme
DdeI

1390

2630

3314

1438

159
2917
1252
1854
192
158
402
198
2279
190
556

190
3002
1752

432

3276
388
444

3287

1256

1756

2998

723

2469

2691

3511
3549

227
3481
21
1940
160
2870
70
2521
237
384
965
544
80
351
1190
383
2868
57
2
3422
1154
44
2111
260
598
1611
169
2850

2368

2492

2779

3464

1529
3500
194

1827

2068

2936

1015

1049

2836

452

1933

2186

2995
271

1354

1778

351
1079
709

1079

2392

3168

1931
159
2917
2
964
1438
268

1963
190
3002
627

2109
432
3549

1371
2392
231
1209
132
2090

3172
2566
2411

3618
3575

198
2176

384
2188

630
2956

2079
2973

1451
2006
1925
2136

1484
2036
1957
2223

1925
2039
1958
2256

1958
2103
1990
2695

2003
2136
2103
2728

327
3625
2451

TB037 12/98

2401

2392
3474
635
220
627

410
1644

1598
1791

2622

3381
414
2332

799
2356

1132
2878

1765
3568

2701

3689
2141
1256

1756

# Sites Locations
10
80
101
1246 1713
DpnI
19
DraI
3
2197 2216
DraIII
1
3474
DrdI
3
1131 1546
DrdII
1
3479
DsaI
2
220
410
EaeI
4
166
223
EagI
1
166
Eam1105I 1
2331
EarI
2
1322 3126
EciI
3
1512 1658
Eco47III 1
692
Eco57I
2
1986 2998
EcoO109I 3
53
402
EcoRI
1
192
EcoRII
4
404
1464
EcoRV
1
217
FauI
8
368
453
1151 3611
FokI
8
657
719
2297 2478
FspI
2
419
2553
GdiII
2
166
2719
HaeI
5
225
409
HaeII
6
611
694
3633
HaeIII
14
HgaI
6
974
1131
3691
HgiEII
1
2024
HhaI
23
Hin4I
2
2330 2404
HincII
1
181
HindIII
1
173
HinfI
9
359
488
1809 2326
HphI
9
483
1058
2817 3023
MaeII
12
MaeIII
15
MboII
8
556
1309
3004 3113
MmeI
3
1653 1837
MnlI
20
MscI
2
225
409
MseI
23
MslI
6
425
620
3101
MspI
18
MspA1I
7
84
267
2025 2966
MwoI
19
NciI
7
448
776
2514 2865
NcoI
1
220
NdeI
1
288
NgoAIV
2
231
3575
NlaIII
15
NlaIV
15
NotI
1
166
NspI
3
783
1075
Pfl1108I 1
2349
PleI
6
367
1332
3417
Psp5II
2
402
444
Psp1406I 4
763
2557
PstI
1
2576
PvuI
1
2701
PvuII
1
1029
RcaI
2
2158 3166

136
2122

544
2288

706
2828

2908

Enzyme
RsaI
SacI
SalI
SapI
Sau96I

# Sites
2
1
1
1
10

Sau3AI
ScaI
ScrFI
SfaNI
SfcI
SspI
StyI

19
1
11
13
5
2
7

TaqII

TfiI
ThaI
TseI
Tsp45I

3
16
26
6

Tsp509I
Tth111I
Tth111II
UbaJI
VspI
XbaI
XhoI
XmnI

11
1
4
12
3
1
1
3

3429

407

2719

2486

444
1585

1598

734
3680
797
2765

920

1141

983

1124

1453
1316

1464
1686

1916
3625

1549

2127

2857

992
3401
1067
3058

1338
3423
2174
3475

1413

2100
3614
3451

2171

2926

1011

2583

2742

1029

1148

1780

1082

1117

1818

1817

2320

3409

2930

3259

2401

Locations
1244 2811
190
179
1322
53
402
2373 2452

444
2469

723
2691

910
3465

2811

352
3135
159
2982
1340
3378
488

1703
3266
180
3507
2679

1894

2572

3693

190

381

1538

2864

3017

3034

992

1413

877
3647

1090

1185

2587

2798

2028

2035

2067

367

2503

996

2930

1183
899
207
326
158
208

Enzymes that do not cut pET-20b(+):

AatII
AflII
AgeI
ApaI
AscI
AvrII
BaeI
BclI
BsmI
BsrGI
BssHII
BstEII
Bsu36I
ClaI
EcoNI
FseI
KpnI
MluI
MunI
NarI
NruI
NsiI
NspV
PacI
PmeI
PmlI
PshAI
RleAI
SacII
SexAI
SfiI
SgfI
SmaI
SnaBI
SpeI
SphI
Sse8387I StuI
SunI
SwaI

1442

Novagen FAX 608-238-1388 E-MAIL novatech@novagen.com

ApaBI
BmgI
BstXI
HpaI
NheI
PflMI
RsrII
SgrAI
SrfI
XcmI