User Protocol TB239VM Rev.

B 0511JN

Page 1 of 2

pET-41a-c(+) Vector
pET-41a(+) DNA (Cat. No. 70556-3) pET-41b(+) DNA (Cat. No. 70557-3) pET-41c(+) DNA (Cat. No. 70558-3) pET-41a(+) sequence landmarks T7 promoter 1167-1183 1166 T7 transcription start GSTTag coding sequence 436-1095 HisTag coding sequence 397-414 STag coding sequence 310-354 Multiple cloning sites (PshA I - Xho I) 175-266 HisTag coding sequence 150-173 T7 terminator 26-72 lacI coding sequence 1574-2656 pBR322 origin 3850 kan coding sequence 4556-5371 F1 origin 5470-5917 Notes All corresponding maps and tables are generated using VectorNTI software. Restriction sites are numbered in accordance with the conventions of the respective software. PshA1 Blunt Cloning Site
PshA1

The pET-41 series is designed for cloning and high-level expression of peptide sequences fused with the 220 aa GSTTag protein. 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). Vector encoded sequence can be completely removed when cloning into the PshAI site (as shown below) and then cleaving the GST fusion protein with Enterokinase.

GAT D

GAC GAC GAC D D D Enterokinase

AAG K

AGT

CCC

PshA1vector
vector GAT GAC GAC D D D insert XXX insert codon

GAC D

AA
K

A/G

pET-41a-c(+) cloning/expression regions

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Ca.

User Protocol TB239VM Rev. B 0511JN

Page 2 of 2 Page

Enzyme AccI AciI AflIII AluI AlwI AlwNI ApaI ApaLI ApoI

# 2 73 3 25 12 1 1 3 7

Locations 197 3559

Enzyme BstYI

# 8

Locations 242 4427 303 4438 1489 5237 2701 2981

Enzyme PstI PvuI PvuII

# 1 1 3 7

Locations 215 4988 2525 232 3595 2618 298 4823 3380 370 522 2072

853

1925

3786

Cac8I ClaI CviJI

43 2 89 11 23 2 1 3 5 1 4 3 4 1 1 17 40 13 13 24 12 46 2 1 18 1 22 1 16 15 1 24 1 31 8 1001 3362 1977 3613 345 3380 393 3499 1955 4128 2525 4373 2265 2295 2776 888 1925 300 2431 198 190 2431 559 5688 3482 183 183 1013 1084 54 236 252 *1543 1060 1060 3670 4900 1358 2795 4900 3894 886 5643 1233 1365 2599 668 1202 4679

RsaI

4202 2136 1905 236 4785 3603 332 5490 4100 2200 5501 4601

DdeI DpnI DraI DraIII DrdI EaeI 4860 2477 2795 3074 EagI EarI EcoNI EcoO109I 381 2564 1309 1247 2694 1323 1268 5725 2136 4643 1382 EcoRI EcoRV FauI Fnu4HI 2410 *2907 FokI HaeII

SacI SacII SalI SapI Sau3AI Sau96I ScaI ScrFI SfaNI SfcI SgrAI SmaI SpeI SphI SspI StuI StyI SwaI TaiI TaqI TfiI

1 1 1 2 23 13 2 22 24 5 1 1 1 1 2 1 4 1 15 19 8

207 394 196 1013 3670

AscI AvaI AvaII AvrII BamHI BanI

1 2 4 1 1 9

217 175 598 132 242 296 1845

370

522

211 1244 4862 425 1400 4913 227 58 668

1167

4051

4242

5907

BanII

207 5763

BbsI BbvI BcgI BclI BfaI

3 24 4 2 10

2071

5480

*177 657 71 2768

1067 1939 133 2803

2251

*3366

HaeIII HgaI

132

222

256

426 4281

1081 4588

1134 5839

HhaI HincII HindIII

BglII BpmI Bpu10I BsaAI BsaBI BsaHI BsaJI BsaWI

1 3 2 2 3 5 12 9

303 *1763 2895 3541 1198 1248 2252 *5005 5688 1208 1269 2986 1383 1882 2565 3316

2604 4955

2839 5127

3343 5218

3761

4899

HinfI HpaI HphI KpnI MaeIII MboII

TseI Tsp45I

24 6 2106 5861 3228 3441 3536 5135

Tsp509I TspRI Tth111I XbaI XcmI XhoI XmnI

29 11 1 1 3 1 3 3534 1133 1781 175 702 3347 5377 2297 2315

3 2978

281 3992 *1776 2710 207 4104

293 4139 *1976 3702 1425

2244 5123 *2949 4126 1909

2747

MluI MnlI MscI

BsgI BsiEI BsiHKAI

4 5 7

*811 186 176 3607

4988 2783

MseI MslI

BslI BsmBI BsmFI Bsp1286I BspEI BsrI BsrBI BsrDI BsrFI

28 3 5 12 2 20 5 3 6 804 *1972 293 5789 1154 2338 1235 *3719 *3608 1244 1611 4942 *5387 *5832 3 2978 2540 274 *3430 1104 5004 1386 *3060 *5903

MspA1I

10

85 2618

MwoI NarI NciI NcoI NdeI NlaIII NlaIV NotI NruI

36 4 12 1 1 29 22 1 1 2 5 3 10 1 266 183 4645 4838 857 322 5104 1400 1507 3134 5251 3426 3790 Enzymes that do not cut pET-41a(+): AatII BseRI NheI SanDI SunI AflII BspMI PacI SexAI UbaEI AhdI Bsu36I PmeI SfiI BglI FseI PmlI SnaBI BsaI FspI RsrII SrfI 256 1095 1248 1269 1383 2565

BsrGI BssHII BssSI Bst1107I BstEII BstXI

1 2 1 1 1 3

230 217 *3959 3559 2106 1727 1856 1979 2336

NsiI NspI PflMI PleI PshAI

*On complementary strand. 2010 EMD Chemicals, Inc., an Affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. Novagen is a registered trademark of Merck, KGaA, Darmstadt, Germany. VectorNTI is a registered trademark of Life Technologies Corporation in the United States and/or certain other countries. The terms and conditions governing EMD Chemicals sale of this product are as indicated on our website (www.merck4biosciences.com).

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