Novagen
TB336 10/02
pACYCDuet-1 Vector
Cat. No. pACYCDuet™-1 is designed for the coexpression of two target genes. The vector
pACYCDuet-1 DNA 71147-3 contains two multiple cloning sites (MCS), each of which is preceded by a T7
pACYCDuet-1 sequence landmarks promoter/lac operator and ribosome binding site (rbs). The vector also carries the P15A
T7 promoter-1 3992–4008 replicon, lacI gene and chloramphenicol resistance gene. This vector can be used in
T7 transcription start-1 1 combination with pETDuet™-1 (Cat. No. 71146-3) in an appropriate host strain for the
His•Tag® coding sequence 83–100 coexpression of up to 4 target genes. Genes inserted into MCS1 can be sequenced using
Multiple cloning sites-1 the ACYCDuetUP1 Primer (Cat. No. 71178-3) and DuetDOWN1 Primer (Cat. No. 71179-
(Nco I–Afl II) 69–168 3). Genes inserted into MCS2 can be sequenced using the DuetUP2 Primer (Cat. No.
T7 promoter-2 214–230 71180-3) and T7 Terminator Primer (Cat. No. 69337-3).
T7 transcription start-2 231
Multiple cloning sites-2 EcoN I (3981)
(Nde I–Avr II) 297–438 MCS1
BsrG I (190) T7lac
S•Tag™ coding sequence 366–410
ApaL I (3533)
T7 terminator 462–509 Mlu I (3513) Bpu1102 I (451) Nco I (69)
Afl III (3513) EcoO109 I (478) His•Tag
P15A origin 1750–2662 Bcl I (3499) BamH I (106)
Bsu36 I (517)
cat (CmR) coding sequence 732–1388 EcoR I (112)
Tth111 I (626) Sac I (122)
lacI coding sequence 2789–3868 BstE II (3331) Drd I (626) BspM I (124)
)
68
Apa I (3310) 38 Asc I (125)
Sse8387 I (135)
9–
Sca I (757) Pst I (135)
278
Sal I (137)
la c I (
Hind III (143)
Not I (150)
Msc I (907) Afl II (163)
732–1388)
Hpa I (3011)
Ava II (2962)
pACYCDuet-1 MCS2
(4008 bp) T7lac
Cm R (
Ehe I (2876) Nde I (298)
Nar I (2875) BspE I (1174) Bgl II (305)
Mun I (311)
EcoR V (319)
NgoA IV I (324)
Fse I (328)
Eco57 I (2653) Pvu I (337)
Xba I (2593) Bpu10 I (1398) Sgf I (337)
Aat II (346)
BsaA I (1481) Kpn I (352)
Xho I (354)
P15 S•Tag
A ori Eco47 III (1751) Pac I (429)
Nsp I (2330) Nhe I (1752) Avr II (433)
BssS I (2128) Sac II (2004) Bst1107 I (1765) T7 terminator
Xmn I (1808)
SgrA I (1838)
ACYCDuetUP1
Primer #71178-3 EcoN I T7 promoter-1
GCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA
T7 transcription start-1
lac operator rbs Nco I His•Tag
GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGGGCAGCAGCCATCACCATCATCACCAC
MetGlySerSerHisHisHisHisHisHis
BspM I BsrG I
Sac I Pst I DuetUP2 Primer
BamH I EcoR I Ecl136 I Asc I Sse8387 I Sal I Hind III Not I Afl II #71180-3
AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCC
SerGlnAspProAsnSerSerSerAlaArgLeuGlnValAspLysLeuAlaAlaAlaEnd
DuetDOWN1 Primer
#71179-3
DuetUP2 Primer T7 transcription start-2
#71180-3 T7 promoter-2 lac operator rbs Nde I
GCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACAT
DuetDOWN Primer
#71179-3
NgoA IV Pvu I Xho I
Nde I Bgl II Mun I EcoR V Fse I Sgf I Aat II Kpn I Ava I S•Tag
ATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAG
MetAlaAspLeuAsnTrpIleSerAlaGlyHisAlaIleAlaAspValGlyThrLeuGluSerGlyLysGluThrAlaAlaAlaLysPheGluArgGln
EcoO109 I
S•Tag Pac I Avr II Bpu1102 I T7 terminator
CACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG
HisMetAspSerSerThrSerAlaAlaEnd T7 Terminator Primer
#69337-3
pACYCDuet-1 cloning/expression regions
Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144
Novagen
pACYCDuet-1 Restriction Sites TB336 10/02
Enzyme # Sites Locations Enzyme # Sites Locations Enzyme # Sites Locations
AatII 1 346 Eco47III 1 1751 Sse8387I 1 135
AccI 3 138 411 1764 Eco57I 1 2653 SspI 2 862 2589
AciI 49 EcoNI 1 3981 StyI 3 69 433 473
AflII 1 163 EcoO109I 1 478 TaiI 7 346 913 1088 1483 1495
AflIII 1 3513 EcoRI 1 112 3783 3856
AluI 18 EcoRII 14 TaqI 15
Alw26I 7 946 1499 2198 2898 3285 EcoRV 1 319 TfiI 2 821 2835
3411 3816 EheI 1 2876 ThaI 22
AlwI 4 101 114 2732 3957 FauI 10 743 1187 2690 2800 2842 TseI 16
AlwNI 2 1706 2354 3009 3317 3704 3771 3796 Tsp45I 4 539 1578 2427 3331
ApaI 1 3310 Fnu4HI 30 Tsp509I 23
ApaLI 1 3533 FokI 4 644 1190 3458 3467 TspRI 14
ApoI 5 112 384 632 644 3238 FseI 1 328 Tth111I 1 626
AscI 1 125 HaeII 4 1753 2878 3121 3902 VspI 5 213 2575 2771 2830 3991
AvaI 1 354 HaeIII 17 XbaI 1 2593
AvaII 1 2962 HgaI 8 1595 1833 2067 3286 3292 XcmI 3 3128 3146 3662
AvrII 1 433 3521 3566 3965 XhoI 1 354
BamHI 1 106 HhaI 26 XmnI 1 1808
BanI 5 348 704 2744 2874 3593 HincII 2 139 3011
BanII 2 122 3310 HindIII 1 143 Enzymes that do not cut pACYCDuet-1:
BbsI 2 3028 3367 HinfI 11 AhdI BglI BsaBI BsaI BspLU11I ClaI
BbvI 16 HpaI 1 3011 DraIII FspI NruI NsiI PmeI PmlI
BcgI 2 162 3193 HphI 16 PshAI Psp5II RcaI RsrII SanDI SapI
BclI 1 3499 KpnI 1 352 SexAI SfiI SmaI SnaBI SpeI SphI
BfaI 5 415 434 462 1753 2594 MaeIII 10 539 1007 1112 1578 1721 SrfI StuI SunI SwaI
BglII 1 305 2294 2427 2449 3331 3854
BpmI 4 1054 1647 3192 3681 MboII 10 900 1565 1974 1985 2574
Bpu10I 1 1398 2608 3028 3367 3538 3883
Bpu1102I 1 451 MluI 1 3513
BsaAI 1 1481 MnlI 18
BsaHI 3 343 2875 3558 MscI 1 907
BsaJI 11 MseI 23
BsaWI 8 551 566 1174 1838 2161 MslI 4 1458 3147 3177 3465
2291 2691 3194 MspA1I 11
BseRI 2 2385 2428 MspI 24
BsgI 3 1819 3468 3668 MunI 1 311
BsiEI 8 153 199 325 337 625 MwoI 24
1909 2278 2734 NarI 1 2875
BsiHKAI 3 122 1663 3537 NciI 9 626 1436 1528 2221 2318
BslI 11 2742 3087 3896 3947
BsmBI 3 946 1499 2898 NcoI 1 69
BsmFI 2 1554 1674 NdeI 1 298
BsmI 2 776 1183 NgoAIV 1 324
Bsp1286I 5 122 707 1663 3310 3537 NheI 1 1752
BspEI 1 1174 NlaIII 15
BspMI 1 124 NlaIV 11
BsrBI 3 13 243 1926 NotI 1 150
BsrDI 3 1157 3106 3472 NspI 1 2330
BsrFI 6 324 566 1838 2161 2394 NspV 2 642 2488
3827 PacI 1 429
BsrGI 1 190 PflMI 4 401 945 1512 3938
BsrI 16 PinAI 3 566 1838 2161
BssHII 2 125 3102 PleI 9 214 365 399 1887 2317
BssSI 1 2128 3084 3880 3967 3992
Bst1107I 1 1765 Psp1406I 2 1085 3853
BstEII 1 3331 PstI 1 135
BstXI 3 3467 3590 3719 PvuI 1 337
BstYI 4 106 305 2737 3949 PvuII 4 1274 1686 2824 2917
Bsu36I 1 517 RsaI 5 192 350 757 1295 3370
Cac8I 22 SacI 1 122
CviJI 61 SacII 1 2004
DdeI 8 262 451 517 950 1398 SalI 1 137
2213 2476 2942 Sau3AI 13
DpnI 13 Sau96I 8 478 1515 1998 2938 2962
DraI 2 915 1254 3306 3307 3652
DrdI 1 626 ScaI 1 757
DsaI 2 69 2001 ScrFI 23
EaeI 7 150 196 322 326 905 SfaNI 10 842 1327 1605 1955 2053
2182 2839 2736 3148 3151 3339 3480
EagI 3 150 196 322 SfcI 4 29 131 226 4004
EarI 2 2621 3896 SgfI 1 337
Ecl136II 1 120 SgrAI 1 1838
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