Novagen

TB418 0804 Rev. A

pET-50b(+) Vector
Cat. No.
71464-3

pET-50b(+) DNA

19671983
1966
18661883
3751859
342359
131315
168212
2673
23743453
4650
53596171
62746721

1. Cordingley, M.G., Register, R.B., Callahan, P.L., Garsky, V.M., and Colonno, R.J. (1989) J. Virol. 63, 50375045.
2. Wang, Q.M., Johnson, R.B., Cox, G.A., Villarreal, E.C., and Loncharich, R.J. (1997) Anal. Biochem. 252, 238245..

Dra III (6491)

Alo I (6457)
Psi I (6363)

7
in (62
orig
1
f
1)

46721)

Spe I (369)
Bgl II (484)
Msc I (616)
BspM I (818)

Nu
s

Ka n

(53

7
61

Blp I (80)

Nco I (944)

51
(37
ag
T

59

pET-50b(+) sequence landmarks

T7 promoter
T7 transcription start
HisTag coding sequence
NusTag coding sequence
HisTag coding sequence
Multiple cloning sites
(SanD I Avr II)
STag coding sequence
T7 terminator
lacI coding sequence
pBR322 ori
Kan coding sequence
f1 origin

The pET-50b(+) vector carries N-terminal NusTag and HisTag coding sequences followed by a
recognition site for the human rhinovirus (HRV) 3C protease. This protease is highly specic for cleavage of the sequence LEVLFQGP (1), and is active at low temperatures (2). pET-50b(+) also contains an
optional C-terminal thrombin recognition site followed by an STag coding sequence. Unique restriction 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 the helper phage will produce virions containing single-stranded DNA that corresponds to the
coding strand. Therefore, single-stranded sequencing should be performed using the AS STag 18mer
Primer (Cat. No. 71262-3).

859)

pET-50b(+)
(6733 bp)

AlwN I (5004)

ori (4

lac operator
T7 promoter

)
650

Pci I (4588)

Nde I (1894)
XbaI (1932)

Sph I (2199)

Sap I (4472)
BstZ17 I (4359)
Tth111 I (4333)

3)
45
3
4
7
23
lacI (
Mlu I (2724)
BstE II (2905)
Apa I (2935) PspOM I (2931)

Hpa I (3230)

T7 Promoter Primer
#69348-3
lac operator
T7 promoter
Xba I
GATGCGTCCGGCGTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGA

NusTag
HisTag
AATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGGCAGCAGCCATCACCACCATCACCATTCTAGTATG...1443bp...ATGGCTGCCCGT
MetGlySerSerHisHisHisHisHisHisSerSerMet...481 aa...MetAlaAlaArg
Acc65 I
Kpn I
Bae I
SanD I
Xma I
NusTag
HisTag
Sac II
Sma I
AATATTTGCTGGTTCGGTGACGAAGCGACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGCTCTTGAAGTCCTCTTTCAGGGACCCGGGTAC
AsnIleCysTrpPheGlyAspGluAlaThrSerGlySerGlyHisHisHisHisHisHisSerAlaAlaLeuGluValLeuPheGlnGlyProGlyTyr
HRV 3C
Eag I
PshA I
EcoICR I
STag
BsrG
I
Sac I
Asc I
Not I
Sal I Hind III
BamH I EcoR I
CAGGATCCGAATTCTGTACAGGCCTTGGCGCGCCCGACGTCCGTCGACAAGCTTGCGGCCGCAGAGCTCGCTCTGGTGCCACGCGGTAGTAAAGAAACC
GlnAspProAsnSerValGlnAlaLeuAlaArgProThrSerValAspLysLeuAlaAlaAlaGluLeuAlaLeuValProArgGlySerLysGluThr
thrombin
STag
Pac I
Avr II
Xho I
GCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCTCTACTTCTGCTGCTCTCGAGGCTTAATTAACCTAGGCTGCTAAACAAAGCCCGAAAGGAAGC
AlaAlaAlaLysPheGluArgGlnHisMetAspSerSerThrSerAlaAlaLeuGluAlaEnd
AS STag 18mer Primer
#71262-3

pET-50b(+) cloning/expression region

Novagen ordering 800-526-7319 technical support 800-207-0144
A Brand of EMD Biosciences, Inc., an Afliate of Merck KGaA, Darmstadt, Germany

Avr II (131)
Pac I (141)
Xho I (147)
STag
EcoICR I (236)
Sac I (238)
Eag I (242)
Not I (242)
Hind III (249)
Sal I (255)
PshA I (262)
Asc I (270)
BsrG I (283)
EcoR I (289)
Bae I (294)
BamH I (295)
Acc65 I (302)
Xma I (306)
Kpn I (306)
Sma I (308)
SanD I (310)
Sac II (338)
HisTag

Novagen

TB418 0804 Rev.A

pET-50b(+) Restriction Sites

Enzyme
AatII
Acc65I
AccI
AclI
AfeI
AIII
AleI
AloI
AlwNI
ApaI
ApaLI
AscI
AseI
AvaI
AvrII
BaeI
BamHI
BanI

# Sites
2
1
3
3
2
2
2
1
1
1
3
1
5
2
1
1
1
10

BanII

BbeI
4
BbsI
4
BceAI
11
BcgI
3
BciVI
4
BclI
2
BfrBI
2
BglII
1
BlpI
2
Bme1580I 4
BmrI
6
BpmI
Bpu10I
BpuEI
BsaAI
BsaBI
BsaHI

3
2
5
2
3
7

BsaWI

BsaXI
BseYI
BsgI
BsiEI

3
4
4
8

BsiHKAI

BsmAI

BsmBI
BsmFI
BsmI
Bsp1286I
BspCNI

3
4
3
13
10

BspEI
BspHI
BspLU11I
BspMI
BsrBI
BsrDI
BsrFI

2
3
1
1
5
4
9

BsrGI
BssHII
BssSI
Bst1107I
BstAPI
BstBI
BstEII

1
5
2
1
3
2
1

Locations
266
1729
302
256
1070
2386 3913
2129 3842
2724 4588
1441 1458
6457
5004
2935
2704 4402
270
1981 3409
147
306
131
294
295
223
302
2181 2644
238
2108
6566
2050 2071
749
2870
236
1815
1474
5638
484
80
2708
1055
4327
2562
3694
21
4340
1997
263
2681
2
4794
331
1649
1497
245
3509
238
3582
2421
5806
3339
296
528
93
3848
2
2122
4588
818
864
614
568
2043
283
270
1160
4359
985
187
2905

3050
3181
2738
5904
819
2935
2253

Enzyme
BstXI
BstYI
4358
6276

4902
3468

5989

991
3363
2122

2046
3493
2935

2185
3209

3367
3706

4165
4791

6185

4406
2650

4906
2887

3051
5807
3538
6491
2007
1726
3364
1117
4941
3399
3123
2575
656
4504
423
4406
2826

3043
5925
6455
3258
2775
794
4928
1315
4906
2952

4229
2185
5674

5806
3859
5751

6706

114
4388
3777
5308

878
4876

3309
5285

1953
995
924
2410

4521
2771
988
5744

6189
3137
1440
6592

1088
4761

1280

1769

1511
1786

2407

6178

2067
6528
5445

3527

4115
4679

4977

5218

3785
2047

2068

2182

3546

3777

4892
3748
1330

1711

2224

2708

3339

4229

3686
5820

6183

6635
2034

3135

# Sites Locations
3
2526 2655
9
295
484
3780 5229
BstZ17I 1
4359
BtgI
4
335
505
BtsI
5
648
3089
ClaI
5
1045 1123
DraIII
1
6491
DrdI
3
4281 4696
EaeI
7
242
512
2164 3398
EagI
1
242
EarI
3
2342 4472
EciI
5
798
1353
Eco57I
2
1367 5136
Eco57MI 5
1367 2562
EcoICRI 1
236
EcoNI
3
318
2259
EcoO109I 4
53
310
EcoRI
1
289
EcoRV
2
452
1043
HaeII
14
HincII
4
257
441
HindIII
1
249
HpaI
1
3230
KasI
4
2046 2067
KpnI
1
306
MluI
1
2724
MscI
1
616
MslI
8
1441 1458
3575 3770
NaeI
2
2036 6594
NarI
4
2047 2068
NcoI
1
944
NdeI
1
1894
NgoMIV 2
2034 6592
NotI
1
242
NruI
4
751
1176
NsiI
2
5640 5906
NspI
4
2199 3933
NspV
2
187
1786
PacI
1
141
PciI
1
4588
PMI
3
179
2306
PfoI
2
2291 4230
PpiI
2
5303 6457
PpuMI
2
310
3594
PshAI
1
262
PsiI
1
6363
PspOMI 1
2931
PvuI
2
794
1711
PvuII
3
3324 3417
SacI
1
238
SacII
1
338
SalI
1
255
SanDI
1
310
SapI
1
4472
SfcI
5
1835 1966
SfoI
4
2048 2069
SgrAI
2
1440 2043
SmaI
1
308
SmlI
6
36
147
5233
SpeI
1
369
SphI
1
2199
SspI
3
398
5715
StuI
2
280
669
StyI
5
57
131
TaqII
5
426
3523
TatI
2
283
4392
TspGWI 9
274
1245
3713 4031

2778
1399
5240

2288
6039

944
3457
1711

2161
5651
2001

5738
5481

6446
614

901

2032

5603
2515

4650

4796

3051

4115

5136

5702
2157

3594

836

3230

2181

3363

2776
4161

3064

2182

3364

1800

5447

4225

4592

3500

Enzyme
Tth111I
XbaI
XcmI
XhoI
XmaI
XmnI
ZraI

# Sites
1
1
3
1
1
3
2

Locations
4333
1932
2580 3096
147
306
1792 4146
264
1727

6053

4179

5044
3365

6710

3517

4694

4956

275
4490

944
6044

1640
6395

1318
5599

1482
5611

1695

6179

Enzymes that do not cut pET-50b(+):

AarI
AII
AgeI
AhdI
BglI
BmgBI BmtI
BplI
BsiWI
Bsu36I BtrI
DraI
FspAI
FspI
MfeI
NheI
PmlI
PsrI
PstI
RsrII
SexAI
SI
SnaBI
SrfI

3094

4853
2183

3114

6283

Novagen ordering 800-526-7319 technical support 800-207-0144

A Brand of EMD Biosciences, Inc., an Afliate of Merck KGaA, Darmstadt, Germany

AsiSI
BsaI
FalI
PinAI
SbfI
Sse8387I

BbvCI
BseRI
FseI
PmeI
ScaI
SwaI