pACYCDuet-1_Vector

pACYCDuet?-1 is designed for the coexpression of two target genes. The vector contains two multiple cloning sites (MCS), each of which is preceded by a T7

promoter/lac operator and ribosome binding site (rbs). The vector also carries the P15A replicon, lacI gene and chloramphenicol resistance gene. This vector can be used in combination with pETDuet?-1 (Cat. No. 71146-3) in an appropriate host strain for the coexpression of up to 4 target genes. Genes inserted into MCS1 can be sequenced using the ACYCDuetUP1 Primer (Cat. No. 71178-3) and DuetDOWN1 Primer (Cat. No. 71179-3). Genes inserted into MCS2 can be sequenced using the DuetUP2 Primer (Cat. No.71180-3) and T7 Terminator Primer (Cat. No. 69337-3).

Novagen ?ORDERING 800-526-7319 ?

TECHNICAL SUPPORT

800-207-0144

G G G G A A T T G T G A G C G G A T A A C A A T T C C C C T G T A G A A A T A A T T T T G T T T A A C T T T A A T A A G G A G A T A T A C C A T G G G C A G C A G C C A T C A C C A T C A T C A C C A C M e t G l y S e r S e r H i s H i s H i s H i s H i s H i s Nco I

His?Tag T7 transcription start-1

lac operator rbs T7 terminator C A C A T G G A C T C G T C T A C T A G C G C A G C T T A A T T A A C C T A G G C T G C T G C C A C C G C T G A G C A A T A A C T A G C A T A A C C C C T T G G G G C C T C T A A A C G G G T C T T G H i s M e t A s p S e r S e r T h r S e r A l a A l a E n d Eco O109 I

Bpu 1102 I

Pac I Avr II T7 Terminator Primer

#69337-3

S?Tag ACYCDuetUP1

Primer #71178-3G C C A T A C C G C G A A A G G T T T T G C G C C A T T C G A T G G T G T C C G G G A T C T C G A C G C T C T C C C T T A T G C G A C T C C T G C A T T A G G A A A T T A A T A C G A C T C A C T A T A Eco N I

T7 promoter-1A G C C A G G A T C C G A A T T C G A G C T C G G C G C G C C T G C A G G T C G A C A A G C T T G C G G C C G C A T A A T G C T T A A G T C G A A C A G A A A G T A A T C G T A T T G T A C A C G G C C S e r G l n A s p P r o A s n S e r S e r S e r A l a A r g L e u G l n V a l A s p L y s L e u A l a A l a A l a E n d

Bam H I Eco R I Sac I

Sse 8387 I Asc I Pst I Ecl 136 I Sal I Bsp M I Not I Hin d III Afl II

DuetUP2 Primer #71180-3DuetDOWN1 Primer

#71179-3

Bsr G I G C A T A A T C G A A A T T A A T A C G A C T C A C T A T A G G G G A A T T G T G A G C G G A T A A C A A T T C C C C A T C T T A G T A T A T T A G T T A A G T A T A A G A A G G A G A T A T A C A T Nde I

T7 transcription start-2

lac operator rbs DuetUP2 Primer #71180-3DuetDOWN Primer

#71179-3

T7 promoter-2A T G G C A G A T C T C A A T T G G A T A T C G G C C G G C C A C G C G A T C G C T G A C G T C G G T A C C C T C G A G T C T G G T A A A G A A A C C G C T G C T G C G A A A T T T G A A C G C C A G M e t A l a A s p L e u A s n T r p I l e S e r A l a G l y H i s A l a I l e A l a A s p V a l G l y T h r L e u G l u S e r G l y L y s G l u T h r A l a A l a A l a L y s P h e G l u A r g G l n Nde I Mun I Fse I

Sgf I Kpn I Xho I Bgl II Eco R V Ngo A IV Pvu I

Aat II Ava I S?Tag TB336 10/02

pACYCDuet-1 Vector

pACYCDuet-1 sequence landmarks

T7 promoter-1

3992–4008T7 transcription start-11

His?Tag ?coding sequence 83–100Multiple cloning sites-1(Nco I–A?II)69–168T7 promoter-2

214–230T7 transcription start-2231Multiple cloning sites-2(Nde I–Avr II)

297–438S?Tag?coding sequence 366–410T7 terminator 462–509P15A origin

1750–2662cat (Cm R ) coding sequence 732–1388lacI coding sequence

2789–3868

BsrG I (190)

EcoN I (3981)

ApaL I (3533)Mlu I (3513)Afl III (3513)Bcl I (3499)

BstE II (3331)Apa I (3310)

Hpa I (3011)Ava II (2962)Ehe I (2876)Nar I (2875)

Eco57 I (2653)

Xba I (2593)

Nsp I (2330)

BssS I (2128)Sac II (2004)

BsaA I (1481)Eco47 III (1751)Nhe I (1752)Bst1107 I (1765)

Xmn I (1808)SgrA I (1838)

Bpu10 I (1398)BspE I (1174)Msc I (907)

Sca I (757)

Tth111 I (626)Drd I (626)

Bsu36 I (517)

Bpu1102 I (451)EcoO109 I (478)T7lac

Nde I (298)

Bgl II (305)

Mun I (311)EcoR V (319)NgoA IV I (324)Fse I (328)Pvu I (337)

Sgf I (337)

Aat II (346)

Kpn I (352)

Xho I (354)S?Tag

Pac I (429)Avr II (433)

T7 terminator MCS2T7lac

Nco I (69)His?Tag

BamH I (106)EcoR I (112)Sac I (122)BspM I (124)Asc I (125)

Sse8387 I (135)Pst I (135)Sal I (137)Hind III (143)Not I (150)Afl II (163)MCS1l a c I (

278

9–

38

68

)

C m R (732–1388)

P 15

A o r i

pACYCDuet-1

(4008 bp)

pACYCDuet-1 cloning/expression regions

Novagen

Cat. No.

pACYCDuet-1 DNA

71147-3Edited by Foxit Reader

Copyright(C) by Foxit Software Company,2005-2008For Evaluation Only.

Enzyme# Sites Locations

AatII1346

AccI31384111764

AciI49

A?II1163

A?III13513

AluI18

Alw26I79461499219828983285

34113816

AlwI410111427323957 AlwNI217062354

ApaI13310

ApaLI13533

ApoI51123846326443238 AscI1125

AvaI1354

AvaII12962

AvrII1433

BamHI1106

BanI5348704274428743593 BanII21223310

BbsI230283367

BbvI16

BcgI21623193

BclI13499

BfaI541543446217532594 BglII1305

BpmI41054164731923681

Bpu10I11398

Bpu1102I1451

BsaAI11481

BsaHI334328753558

BsaJI11

BsaWI8551566117418382161

229126913194

BseRI223852428

BsgI3181934683668

BsiEI8153199325337625

190922782734

BsiHKAI312216633537

BslI11

BsmBI394614992898

BsmFI215541674

BsmI27761183

Bsp1286I5122707166333103537 BspEI11174

BspMI1124

BsrBI3132431926

BsrDI3115731063472

BsrFI6324566183821612394

3827

BsrGI1190

BsrI16

BssHII21253102

BssSI12128

Bst1107I11765

BstEII13331

BstXI3346735903719

BstYI410630527373949

Bsu36I1517

Cac8I22

CviJI61

DdeI82624515179501398

221324762942

DpnI13

DraI29151254

DrdI1626

DsaI2692001

EaeI7150196322326905

21822839

EagI3150196322

EarI226213896

Ecl136II1120Enzyme# Sites Locations

Eco47III11751

Eco57I12653

EcoNI13981

EcoO109I1478

EcoRI1112

EcoRII14

EcoRV1319

EheI12876

FauI107431187269028002842

30093317370437713796

Fnu4HI30

FokI4644119034583467

FseI1328

HaeII41753287831213902

HaeIII17

HgaI815951833206732863292

352135663965

HhaI26

HincII21393011

HindIII1143

HinfI11

HpaI13011

HphI16

KpnI1352

MaeIII105391007111215781721

22942427244933313854

MboII109001565197419852574

26083028336735383883

MluI13513

MnlI18

MscI1907

MseI23

MslI41458314731773465

MspA1I11

MspI24

MunI1311

MwoI24

NarI12875

NciI96261436152822212318

2742308738963947

NcoI169

NdeI1298

NgoAIV1324

NheI11752

NlaIII15

NlaIV11

NotI1150

NspI12330

NspV26422488

PacI1429

P?MI440194515123938

PinAI356618382161

PleI921436539918872317

3084388039673992

Psp1406I210853853

PstI1135

PvuI1337

PvuII41274168628242917

RsaI519235075712953370

SacI1122

SacII12004

SalI1137

Sau3AI13

Sau96I84781515199829382962

330633073652

ScaI1757

ScrFI23

SfaNI108421327160519552053

27363148315133393480

SfcI4291312264004

SgfI1337

SgrAI11838

Enzyme# Sites Locations

Sse8387I1135

SspI28622589

StyI369433473

TaiI7346913108814831495

37833856

TaqI15

T?I28212835

ThaI22

TseI16

Tsp45I4539157824273331

Tsp509I23

TspRI14

Tth111I1626

VspI52132575277128303991

XbaI12593

XcmI3312831463662

XhoI1354

XmnI11808

Enzymes that do not cut pACYCDuet-1:

AhdI BglI BsaBI BsaI BspLU11I ClaI

DraIII FspI NruI NsiI PmeI PmlI

PshAI Psp5II RcaI RsrII SanDI SapI

SexAI S?I SmaI SnaBI SpeI SphI

SrfI StuI SunI SwaI

pACYCDuet-1 Restriction Sites TB336 10/02

Novagen ?FAX608-238-1388 ?E-MAIL novatech@https://www.360docs.net/doc/2117382738.html,

Novagen