Map - EMBL

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Map - EMBL
pSP73 Vector
Sequence and Map
Technical Bulletin No. 041
INSTRUCTIONS FOR USE OF PRODUCT P2221. PLEASE DISCARD PREVIOUS VERSIONS.
All technical literature is available on the Internet at www.promega.com
Please visit the web site to verify that you are using the most current version of this Technical Bulletin.
I.
Description ............................................................................................................1
II.
Product Components ...........................................................................................1
III.
pSP73 Vector Multiple Cloning Site and Circle Map ..........................................2
IV.
pSP73 Vector Restriction Sites............................................................................4
V.
pSP73 Vector Sequence .......................................................................................5
VI.
Related Products ..................................................................................................7
VII.
Reference ..............................................................................................................7
I.
Description
The pSP73 Vector(a) (1) offers a wide range of restriction sites, providing greater versatility in cloning and transcription of RNA in vitro. The pSP73 Vector contains the SP6
and T7 RNA polymerase promoters and a unique multiple cloning region which
includes restriction sites for Bgl II, EcoR V, Cla I, EcoR I, Sac I, Kpn I, Sma I, BamH I,
Xba I, Acc I, Sal I, Pst I, Sph I, Hind III, Pvu II and Xho I.
The sequences of Promega vectors are available online at www.promega.com/vectors/
and are also available from the GenBank® database.
II.
Product Components
Product
pSP73 Vector
Size
20µg
Cat.#
P2221
AF9TB041
0100TB041
Storage Conditions: Store the pSP73 Vector at –20°C.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/00
Part# TB041
Page 1
III. pSP73 Vector Multiple Cloning Site and Circle Map
SP6 Transcription Start
5′ . . . ATTTA GGTGA CACTA TAGAA CCAGA TCTGA TATCA TCGAT
SP6 Promoter
Bgl II
Eco R V
Cla I
GAATT CGAGC TCGGT ACCCG GGGAT CCTCT AGAGT CGACC TGCAG
EcoR I
Sac I
Kpn I
Sma I
Bam H I
Xba I
Sal I
Acc I
Pst I
T7 Transcription Start
GCATG CAAGC TTCAG CTGCT CGAGG CCGGT CTCCC TATAG TGAGT CGTAT TA . . . 3′
T7 Promoter
Sph I
Hind III
Pvu II
Xho I
Figure 1. pSP73 Vector promoter and multiple cloning site sequence. The sequence
shown corresponds to RNA synthesized by SP6 RNA polymerase and is complementary to
RNA synthesized by T7 RNA polymerase.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TB041
Page 2
Printed in USA.
Revised 1/00
!
Xmn I 1809
Sca I 1690
Ssp I 2014
Fsp I 1432
Aat II 2132
Nde I
2381
Amp r
Bgl I 1330
pSP73
Vector
(2464bp)
Hpa I
138
SP6
Bgl II
Eco R V
Cla I
Eco R I
Sac I
Kpn I
Sma I
Bam H I
Xba I
Sal I
Acc I
Pst I
Sph I
Hind III
Pvu II
Xho I
➞
ori
➞
Pvu I 1580
T7
1 start
6
14
19
24
34
40
42
45
51
57
58
67
73
75
83
87
103
PROMEGA’s
... pSP72 and pSP73
Vectors are identical
except for the orientation
of the multiple cloning
region.
BLUE/WHITE
... screening of recombinants is not possible with
the pSP73 Vector.
Figure 2. pSP73 Vector circle map and sequence reference points.
pSP73 Vector sequence reference points.
SP6 RNA polymerase transcription initiation site
T7 RNA polymerase transcription initiation site
SP6 RNA polymerase promoter (–17 to +3)
T7 RNA polymerase promoter (–17 to +3)
multiple cloning region
β-lactamase coding region
1
103
2448–3
107–120
6–92
1137–1997
Specialized application of the pSP73 Vector.
•
Transcription in vitro from dual opposed promoters (For protocol information, please
request Promega’s Riboprobe® in vitro Transcription Systems(b) Technical Manual,
#TM016).
Note: All Promega technical literature is available
on the Internet at
www.promega.com.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/00
Part# TB041
Page 3
IV. pSP73 Vector Restriction Sites
The following restriction enzyme tables were constructed using DNASTAR® sequence analysis software. Please note that we have not verified this information by restriction digestion with each enzyme
listed. The location given specifies the 3´ end of the cut DNA (the base to the left of the cut site). For
more information on the cut sites of these enzymes, or if you identify a discrepancy, please contact
your local Promega Branch or Distributor. In the U.S., contact Promega Technical Services at 800356-9526. Vector sequences are also available in the GenBank® database (GenBank®/EMBL
Accession Number X65333) and on the Internet at www.promega.com/vectors/.
Table 1. Restriction Enzymes That Cut the pSP73 Vector Between 1 and 5 Times.
Enzyme
Aat II
Acc I
Acc 65 I
Acy I
Afl III
Alw26 I
# of Sites
1
1
1
2
1
5
Alw44 I
Alw N I
AspH I
3
1
5
Ava I
Ava II
BamH I
Ban I
Ban II
Bbu I
Bgl I
Bgl II
Bsa I
BsaO I
BsaH I
BsaJ I
Bsp1286 I
2
2
1
2
1
1
1
1
2
4
2
3
5
BspH I
BspM I
BssS I
BstO I
Cfr10 I
Cla I
Dra I
Dra II
Drd I
Eae I
Ear I
Ecl HK I
EcoICR I
EcoR I
3
2
3
4
2
1
3
1
2
2
2
2
1
1
Location
2132
58
36
1747, 2129
317
102, 1271, 2047, 2200
2242
631, 1877, 2374
733
34, 635, 1796, 1881,
2378
40, 87
1348, 1570
45
36, 1158
34
73
1330
6
102, 1271
233, 657, 1580, 1729
1747, 2129
40, 41, 477
34, 635, 1796, 1881,
2378
1037, 2045, 2150
70, 149
490, 1874, 2181
133, 345, 466, 479
93, 1290
19
1076, 1095, 1787
2186
425, 2294
156, 1598
201, 2005
1210, 2391
32
24
Enzyme # of Sites
EcoR V
1
Fok I
4
Fsp I
1
Hae II
2
Hga I
4
Hinc II
2
Hind II
2
Hind III
1
Hpa I
1
Hsp92 I
2
Kpn I
1
Mae I
4
Mae II
4
MspA1 I
5
Nde I
Nsp I
Ple I
PspA I
Pst I
Pvu I
Pvu II
Rsa I
Sac I
Sal I
Sau 96 I
1
3
5
1
1
1
1
3
1
1
5
Sca I
Sin I
Sma I
Sph I
Sse8387 I
Ssp I
Tfi I
Vsp I
Xba I
Xho I
Xma I
Xmn I
1
2
1
1
1
1
2
4
1
1
1
1
Location
14
1176, 1357, 1644, 2287
1432
195, 565
428, 1006, 1736, 2294
59, 138
59, 138
75
138
1747, 2129
40
52, 812, 1065, 1400
1020, 1436, 1809, 2129
83, 659, 904, 1845,
2311
2381
73, 321, 2238
63, 118, 211, 696, 1199
40
67
1580
83
38, 1690, 2366
34
57
1252, 1331, 1348, 1570
2186
1690
1348, 1570
42
73
67
2014
152, 292
118, 147, 1382, 2415
51
87
40
1809
Note: The enzymes listed in boldface type are available from Promega.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TB041
Page 4
Printed in USA.
Revised 1/00
Table 2. Restriction Enzymes That Do Not Cut the pSP73 Vector.
Acc B7 I
Acc III
Afl II
Age I
Apa I
Asc I
Avr II
Bal I
Bbe I
BbrP I
Bbs I
Bcl I
Blp I
Bpu 1102 I
BsaA I
BsaB I
BsaM I
Bsm I
Bsp120 I
Bsr BR I
BsrG I
BssH II
Bst1107 I
Bst 98 I
Bst E II
Bst X I
Bst Z I
Bsu36 I
Csp I
Csp45 I
Dra III
Dsa I
Eag I
Eco47 III
Eco52 I
Eco72 I
Eco81 I
EcoN I
Ehe I
Fse I
I-Ppo I
Kas I
Mlu I
Nae I
Nar I
Nco I
NgoM IV
Nhe I
Not I
Nru I
Nsi I
Pac I
PaeR7 I
Pfl M I
PinA I
Pme I
Pml I
Ppu10 I
PpuM I
PshA I
Psp5 II
Rsr II
Sac II
Sfi I
Sgf I(c)
SgrA I
SnaB I
Spe I
Spl I
Srf I
Stu I
Sty I
Swa I
Tth111 I
Xcm I
Note: The enzymes listed in boldface type are available from Promega.
Table 3. Restriction Enzymes That Cut the pSP73 Vector 6 or More Times.
Aci I
Alu I
Bbv I
Bsr I
Bsr S I
Bst 71 I
BstU I
Cfo I
Dde I
Dpn I
Dpn II
Fnu4H I
Hae III
Hha I
Hinf I
Hpa II
Hph I
Hsp92 II
Mae III
Mbo I
Mbo II
Mnl I
Mse I
Msp I
Nci I
Nde II
Nla III
Nla IV
Sau3A I
ScrF I
SfaN I
Taq I
Tru9 I
Xho II
Note: The enzymes listed in boldface type are available from Promega.
V.
pSP73 Vector Sequence
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
GAACCAGATC
TCTAGAGTCG
CCCTATAGTG
TGAATCGGCC
CGCTTCCTCG
CGGTATCAGC
GATAACGCAG
CCGTAAAAAG
ACGAGCATCA
GGACTATAAA
TCCTGTTCCG
CGGGAAGCGT
GTGTAGGTCG
GCCCGACCGC
TAAGACACGA
AGAGCGAGGT
TGATATCATC
ACCTGCAGGC
AGTCGTATTA
AACGCGCGGG
CTCACTGACT
TCACTCAAAG
GAAAGAACAT
GCCGCGTTGC
CAAAAATCGA
GATACCAGGC
ACCCTGCCGC
GGCGCTTTCT
TTCGCTCCAA
TGCGCCTTAT
CTTATCGCCA
ATGTAGGCGG
GATGAATTCG
ATGCAAGCTT
ATTTCGATAA
GAGAGGCGGT
CGCTGCGCTC
GCGGTAATAC
GTGAGCAAAA
TGGCGTTTTT
CGCTCAAGTC
GTTTCCCCCT
TTACCGGATA
CATAGCTCAC
GCTGGGCTGT
CCGGTAACTA
CTGGCAGCAG
TGCTACAGAG
AGCTCGGTAC
CAGCTGCTCG
GCCAGGTTAA
TTGCGTATTG
GGTCGTTCGG
GGTTATCCAC
GGCCAGCAAA
CCATAGGCTC
AGAGGTGGCG
GGAAGCTCCC
CCTGTCCGCC
GCTGTAGGTA
GTGCACGAAC
TCGTCTTGAG
CCACTGGTAA
TTCTTGAAGT
CCGGGGATCC
AGGCCGGTCT
CCTGCATTAA
GGCGCTCTTC
CTGCGGCGAG
AGAATCAGGG
AGGCCAGGAA
CGCCCCCCTG
AAACCCGACA
TCGTGCGCTC
TTTCTCCCTT
TCTCAGTTCG
CCCCCGTTCA
TCCAACCCGG
CAGGATTAGC
GGTGGCCTAA
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/00
Part# TB041
Page 5
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
CTACGGCTAC
CAGTTACCTT
ACCGCTGGTA
AAAAAAAGGA
CTCAGTGGAA
AAAAGGATCT
AATCTAAAGT
TCAGTGAGGC
GCCTGACTCC
TGGCCCCAGT
ATTTATCAGC
CCTGCAACTT
TAGAGTAAGT
CTACAGGCAT
TCCGGTTCCC
AAAAGCGGTT
CCGCAGTGTT
GTCATGCCAT
GTCATTCTGA
CAATACGGGA
ATTGGAAAAC
GAGATCCAGT
CTTTTACTTT
GCCGCAAAAA
CTTCCTTTTT
GCGGATACAT
CGCACATTTC
CATGACATTA
CGCGTTTCGG
ACGGTCACAG
GGGCGCGTCA
CATCAGAGCA
GAACGCGGCT
TAGGTGACAC
ACTAGAAGAA
CGGAAAAAGA
GCGGTGGTTT
TCTCAAGAAG
CGAAAACTCA
TCACCTAGAT
ATATATGAGT
ACCTATCTCA
CCGTCGTGTA
GCTGCAATGA
AATAAACCAG
TATCCGCCTC
AGTTCGCCAG
CGTGGTGTCA
AACGATCAAG
AGCTCCTTCG
ATCACTCATG
CCGTAAGATG
GAATAGTGTA
TAATACCGCG
GTTCTTCGGG
TCGATGTAAC
CACCAGCGTT
AGGGAATAAG
CAATATTATT
ATTTGAATGT
CCCGAAAAGT
ACCTATAAAA
TGATGACGGT
CTTGTCTGTA
GCGGGTGTTG
GATTGTACTG
ACAATTAATA
TATA
CAGTATTTGG
GTTGGTAGCT
TTTTGTTTGC
ATCCTTTGAT
CGTTAAGGGA
CCTTTTAAAT
AAACTTGGTC
GCGATCTGTC
GATAACTACG
TACCGCGAGA
CCAGCCGGAA
CATCCAGTCT
TTAATAGTTT
CGCTCGTCGT
GCGAGTTACA
GTCCTCCGAT
GTTATGGCAG
CTTTTCTGTG
TGCGGCGACC
CCACATAGCA
GCGAAAACTC
CCACTCGTGC
TCTGGGTGAG
GGCGACACGG
GAAGCATTTA
ATTTAGAAAA
GCCACCTGAC
ATAGGCGTAT
GAAAACCTCT
AGCGGATGCC
GCGGGTGTCG
AGAGTGCACC
CATAACCTTA
TATCTGCGCT
CTTGATCCGG
AAGCAGCAGA
CTTTTCTACG
TTTTGGTCAT
TAAAAATGAA
TGACAGTTAC
TATTTCGTTC
ATACGGGAGG
CCCACGCTCA
GGGCCGAGCG
ATTAATTGTT
GCGCAACGTT
TTGGTATGGC
TGATCCCCCA
CGTTGTCAGA
CACTGCATAA
ACTGGTGAGT
GAGTTGCTCT
GAACTTTAAA
TCAAGGATCT
ACCCAACTGA
CAAAAACAGG
AAATGTTGAA
TCAGGGTTAT
ATAAACAAAT
GTCTAAGAAA
CACGAGGCCC
GACACATGCA
GGGAGCAGAC
GGGCTGGCTT
ATATGGACAT
TGTATCATAC
CTGCTGAAGC
CAAACAAACC
TTACGCGCAG
GGGTCTGACG
GAGATTATCA
GTTTTAAATC
CAATGCTTAA
ATCCATAGTT
GCTTACCATC
CCGGCTCCAG
CAGAAGTGGT
GCCGGGAAGC
GTTGCCATTG
TTCATTCAGC
TGTTGTGCAA
AGTAAGTTGG
TTCTCTTACT
ACTCAACCAA
TGCCCGGCGT
AGTGCTCATC
TACCGCTGTT
TCTTCAGCAT
AAGGCAAAAT
TACTCATACT
TGTCTCATGA
AGGGGTTCCG
CCATTATTAT
TTTCGTCTCG
GCTCCCGGAG
AAGCCCGTCA
AACTATGCGG
ATTGTCGTTA
ACATACGATT
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TB041
Page 6
Printed in USA.
Revised 1/00
VI. Related Products
Product
pSP64 Poly(A) Vector
pSP72 Vector(a)
Size
20µg
20µg
Cat.#
P1241
P2191
Product
pGEM®-3Z Vector(a)
pGEM®-4Z Vector(a)
pGEM®-3Zf(+) Vector(a)
pGEM®-3Zf(−) Vector(a)
pGEM®-5Zf(+) Vector(a)
pGEM®-5Zf(−) Vector(a)
pGEM®-7Zf(+) Vector(a)
pGEM®-7Zf(−) Vector(a)
pGEM®-9Zf(−) Vector(a)
pGEM®-11Zf(+) Vector(a)
pGEM®-11Zf(−) Vector(a)
pGEM®-13Zf(+) Vector(a)
Size
20µg
20µg
20µg
20µg
20µg
20µg
20µg
20µg
20µg
20µg
20µg
20µg
Cat.#
P2151
P2161
P2271
P2261
P2241
P2351
P2251
P2371
P2391
P2411
P2421
P2541
All pGEM® Vectors are provided with a glycerol stock of bacterial strain JM109. The JM109 cells do not contain
vector and are not competent.
Sequencing Primers
Product
SP6 Promoter Primer
T7 Promoter Primer
pUC/M13 Primer, Reverse (17mer)
pUC/M13 Primer, Forward (17mer)
pUC/M13 Primer, Forward (24mer)
pUC/M13 Primer, Reverse (22mer)
Size
2µg
2µg
2µg
2µg
2µg
2µg
Cat.#
Q5011
Q5021
Q5401
Q5391
Q5601
Q5421
Riboprobe® in vitro Transcription Systems
Product
Riboprobe® System - SP6(b)
Riboprobe® System - T7(b)
Cat.#
P1420
P1440
VII. Reference
1. Krieg, P.A. and Melton, D.A. (1987) In vitro RNA synthesis with SP6 RNA polymerase. Meth.
Enzymol. 155, 397.
(a)U.S. Pat. No. 4,766,072.
(b)U.S. Pat. No. 5,552,302
and other patents. Inhibitors of Angiogenin, which comprises a segment of human PRI, is the subject of U.S. Pat. No.
4,966,964 and other patents assigned to the President and Fellows of Harvard College and exclusively licensed to Promega Corporation.
(c)U.S. Pat. No. 5,391,487 has been issued to Promega Corporation for Restriction Endonuclease Sgf I.
© 1988–1999 Promega Corporation. All Rights Reserved.
pGEM and RiboProbe are trademarks of Promega Corporation and are registered with the U.S.
Patent and Trademark Office. DNAStar is a registered trademark of DNAStar, Inc. GenBank is a registered trademark of the U.S. Department of Health and Human Services.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the
Promega online catalog for the most up-to-date information on Promega products.
Promega Corporation
2800 Woods Hollow Road
Madison, WI 53711-5399
USA
Telephone
608-274-4330
Fax
608-277-2516
Internet
www.promega.com
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/00
Part# TB041
Page 7

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