pGEM-3Zf(+) Vector

Technical Bulletin No. 086

INSTRUCTIONS FOR USE OF PRODUCT P2271. PLEASE DISCARD PREVIOUS VERSIONS.
All Technical Literature is Available on the Internet at www.promega.com

I.

Description ............................................................................................................1

II.

Product Components ...........................................................................................2

III.

Multiple Cloning Site and Circle Map..................................................................2

IV.

pGEM-3Zf(+) Vector Restriction Sites ...............................................................4

V.

pGEM-3Zf(+) Vector Sequence ..........................................................................5

VI.

Related Products ..................................................................................................7

VII.

Reference ..............................................................................................................8

I.

Description
The pGEM-3Zf(+) Vector(a) is a derivative of the pGEM-3Z Vector and contains
the origin of replication of the filamentous phage f1. The plasmid serves as a standard cloning vector, as a template for in vitro transcription and as a template for the
production of circular ssDNA.
The pGEM-3Zf(+) Vector contains SP6 and T7 RNA polymerase promoters flanking
a multiple cloning site within the -peptide coding region of -galactosidase (1).
Insertional inactivation of the -peptide allows recombinant clones to be directly
identified by color screening on indicator plates. The multiple cloning region is
unique and includes restriction sites for EcoR I, Sac I, Kpn I, Ava I, Sma I, BamH I,
Xba I, Sal I, Acc I, Hinc II, Pst I, Sph I, and Hind III.

AF9TB086

The sequences of Promega's Vectors are available online at

www.promega.com/vectors and are also available from the GenBank database.
The GenBank accession number is X65306.

0103TB086

For induction of ssDNA, bacterial cells with the F episome (e.g., JM109, XL-1 Blue,
DH5) containing pGEM-3Zf(+) Vector recombinants are infected with an appropriate helper phage. The plasmid then enters the f1 replication mode and the resulting ssDNA is exported from the cell as an encapsidated virus-like particle. The
sequence of the ssDNA rescued upon infection with helper phage is complementary
to the sequence shown in Figure 1. The exported ssDNA can be used for mutagenesis in vitro or can be sequenced using Promega's T7 Promoter Primer (Cat.# Q5021)
or pUC/M13 Forward Primer (Cat.# Q5601).

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 1/03

Part# TB086
Page 1

II.

Product Components
Product
pGEM-3Zf(+) Vector

Size
20g

Cat.#
P2271

The pGEM-3Zf(+) Vector is provided with a glycerol stock of bacterial strain JM109. The
JM109 cells do not contain vector and are not competent cells.

Storage Conditions: Store the pGEM-3Zf(+) Vector at 20C and the glycerol
stock of JM109 cells at 70C.
III. Multiple Cloning Site and Circle Map

T7 Transcription Start
5 . . . TGTAA TACGA CTCAC TATAG GGCGA ATTCG AGCTC GGTAC CCGGG GATCC
T7 Promoter
EcoR I

Sac I

Kpn I

Ava I
Sma I

BamH I

TCTAG AGTCG ACCTG CAGGC ATGCA AGCTT GAGTA TTCTA TAGTG TCACC TAAAT . . . 3
SP6 Promoter
Xba I

Sal I
Acc I
Hinc II

Pst I

Sph I

Hind III

0283MA06/9A

SP6 Transcription Start

Figure 1. pGEM-3Zf(+) Vector promoter and multiple cloning site sequence. The
sequence shown corresponds to RNA synthesized by T7 RNA polymerase and is complementary to RNA synthesized by SP6 RNA polymerase. The strand shown is complementary
to the ssDNA strand produced by this vector.

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# TB086
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Printed in USA.
Revised 1/03

Nde I
2509

Sca I 1818
Amp r

pGEM -3Zf(+)
Vector

lacZ

(3197bp)

ori

Eco R I
Sac I
Kpn I
Ava I
Sma I
BamH I
Xba I
Sal I
Acc I
Hinc II
Pst I
Sph I
Hind III

f1 ori

T7

Xmn I 1937

1 start
5
15
21
21
23
26
32
38
39
40
48
54
56
69

SP6

0282VC02/5A

Aat II
2260

Figure 2. pGEM-3Zf(+) Vector circle map and sequence reference points.

Sequence reference points:
T7 RNA polymerase transcription initiation site
SP6 RNA polymerase transcription initiation site
T7 RNA polymerase promoter (17 to +3)
SP6 RNA polymerase promoter (17 to +3)
multiple cloning region
lacZ start codon
lac operon sequences
lac operator
-lactamase (Ampr) coding region
phage f1 region
binding site of pUC/M13 Forward Sequencing Primer
binding site of pUC/M13 Reverse Sequencing Primer

1
69
31813
6786
561
108
30183178; 94323
128144
12652125
25623017
31383154
104120

Specialized applications:
ssDNA production
blue/white screening for recombinants
transcription in vitro from dual opposed promoters (For protocol information, please
request Promegas Riboprobe in vitro Transcription Systems Technical Manual,
#TM016.)

USE the T7
Promoter Primer (Cat.#
Q5021) or pUC/M13
Forward Primer (Cat.#
Q5601) to sequence
ssDNA produced by the
pGEM-3Zf(+) Vector.

Note: The pGEM-3Zf(+)

and pGEM-3Zf()
Vectors are identical
except for the orientation
of the f1 origin.

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 1/03

Part# TB086
Page 3

IV. pGEM-3Zf(+) 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
800-356-9526. Vector sequences are also available on the Internet at www.promega.com/vectors
and from GenBank (GenBank/EMBL Accession Number X65306).
Table 1. Restriction Enzymes That Cut the pGEM-3Zf(+) Vector Between 1 and 5 Times.
Enzyme
Aat II
Acc I
Acc 65 I
Acy I
Afl III
Alw 26 I

# of Sites
1
1
1
2
1
4

Alw 44 I
Alw N I
Asp H I

3
1
5

Ava I
Ava II
BamH I
Ban I
Ban II
Bbu I
Bgl I
Bsa I
Bsa O I

1
2
1
4
2
1
2
1
5

BsaA I
BsaH I
BsaJ I

1
2
5

Bsp H I
Bsp M I
Bss S I
Bst O I

3
1
3
5

Cfr10 I
Dra I
Dra II
Dra III
Drd I
Eae I
Ear I
Ecl HK I
Eco ICR I

2
3
1
1
3
3
3
1
1

Location
2260
39
17
1875, 2257
445
1399, 2175, 2328,
2370
759, 2005, 2502
861
15, 763, 1924, 2009,
2506
21
1476, 1698
26
17, 189, 1286, 2823
15, 2861
54
1458, 3030
1399
361, 785, 1708,
1857, 3058
2786
1875, 2257
21, 22, 184, 605,
3133
1165, 2173, 2278
51
618, 2002, 2309
185, 473, 594, 607,
3134
1418, 2887
1204, 1223, 1915
2314
2786
553, 2422, 2741
284, 1726, 3167
329, 2133, 3075
1338
13

Enzyme
Eco R I
Fok I

# of Sites
1
5

Fsp I
Hae II
Hga I

2
4
5

Hin c II
Hind II
Hin d III
Hsp 92 I
Kpn I
Mae I

1
1
1
2
1
5

Nae I
Nde I
NgoM IV
Nsp I
PspA I
Pst I
Pvu I
Pvu II
Rsa I
Sac I
Sal I
Sca I
Sin I
Sma I
Sph I
Sse 8387 I
Ssp I
Taq I

1
1
1
3
1
1
2
2
3
1
1
1
2
1
1
1
2
5

Tfi I
Vsp I
Xba I
Xma I
Xmn I

2
3
1
1
1

Location
5
1304, 1485, 1772,
2415, 3113
1560, 3037
323, 693, 2937, 2945
556, 1134, 1864,
2422, 3003
40
40
56
1875, 2257
21
33, 940, 1193, 1528,
2937
2889
2509
2887
54, 449, 2366
21
48
1708, 3058
269, 3087
19, 1818, 2494
15
38
1818
1476, 1698
23
54
48
2142, 2578
9, 39, 545, 1989,
2819
280, 420
216, 275, 1510
32
21
1937

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# TB086
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Printed in USA.
Revised 1/03

Table 2. Restriction Enzymes That Do Not Cut the pGEM-3Zf(+) Vector.

Acc B7 I
Acc III
Afl II
Age I
Apa I
Asc I
Avr II
Bal I
Bbe I
Bbr P I
Bbs I
Bcl I
Bgl II

Blp I
Bpu1102 I
BsaB I
BsaM I
Bsm I
Bsp120 I
Bsr BR I
Bsr G I
BssH II
Bst1107 I
Bst 98 I
Bst E II
Bst X I

Bst Z I
Bsu 36 I
Cla I
Csp I
Csp 45 I
Dsa I
Eag I
Eco 47 III
Eco52 I
Eco72 I
Eco81 I
Eco N I
EcoR V

Ehe I
Fse I
Hpa I
I-Ppo I
Kas I
Mlu I
Nar I
Nco I
Nhe I
Not I
Nru I
Nsi I
PaeR7 I

Pac 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

SgrA I
Sna B I
Spe I
Spl I
Srf I
Stu I
Sty I
Swa I
Tth111 I
Xcm I
Xho I

Table 3. Restriction Enzymes That Cut the pGEM-3Zf(+) Vector 6 or More Times.

Aci I
Alu I
Bbv I
Bsp1286 I
Bsr I
BsrS I
Bst71 I

Bst U I
Cfo I
Dde I
Dpn I
Dpn II
Fnu4H I
Hae III

Hha I
Hinf I
Hpa II
Hph I
Hsp 92 II
Mae II
Mae III

Mbo I
Mbo II
Mnl I
Mse I
Msp I
Msp A1 I
Nci I

Nde II
Nla III
Nla IV
Ple I
Sau 3A I
Sau 96 I
ScrF I

SfaN I
Tru 9 I
Xho II

Note: The enzymes listed in boldface type are available from Promega.

V.

pGEM-3Zf(+) Vector Sequence

The sequence shown corresponds to RNA synthesized by T7 RNA polymerase and is complementary to RNA
synthesized by SP6 RNA polymerase. The strand shown is complementary to the ssDNA strand produced by
this vector. The GenBank/EMBL Accession Number is X65306.

1
51
101
151
201
251
301
351
401
451
501
551

GGGCGAATTC
CATGCAAGCT
ATGGTCATAG
ACAACATACG
GTGAGCTAAC
GGGAAACCTG
GAGGCGGTTT
CTGCGCTCGG
GGTAATACGG
GAGCAAAAGG
GCGTTTTTCC
CTCAAGTCAG

GAGCTCGGTA
TGAGTATTCT
CTGTTTCCTG
AGCCGGAAGC
TCACATTAAT
TCGTGCCAGC
GCGTATTGGG
TCGTTCGGCT
TTATCCACAG
CCAGCAAAAG
ATAGGCTCCG
AGGTGGCGAA

CCCGGGGATC
ATAGTGTCAC
TGTGAAATTG
ATAAAGTGTA
TGCGTTGCGC
TGCATTAATG
CGCTCTTCCG
GCGGCGAGCG
AATCAGGGGA
GCCAGGAACC
CCCCCCTGAC
ACCCGACAGG

CTCTAGAGTC
CTAAATAGCT
TTATCCGCTC
AAGCCTGGGG
TCACTGCCCG
AATCGGCCAA
CTTCCTCGCT
GTATCAGCTC
TAACGCAGGA
GTAAAAAGGC
GAGCATCACA
ACTATAAAGA

GACCTGCAGG
TGGCGTAATC
ACAATTCCAC
TGCCTAATGA
CTTTCCAGTC
CGCGCGGGGA
CACTGACTCG
ACTCAAAGGC
AAGAACATGT
CGCGTTGCTG
AAAATCGACG
TACCAGGCGT

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 1/03

Part# TB086
Page 5

601
651
701
751
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
2501

TTCCCCCTGG
ACCGGATACC
TAGCTCACGC
TGGGCTGTGT
GGTAACTATC
GGCAGCAGCC
CTACAGAGTT
GTATTTGGTA
TGGTAGCTCT
TTGTTTGCAA
CCTTTGATCT
TTAAGGGATT
TTTTAAATTA
ACTTGGTCTG
GATCTGTCTA
TAACTACGAT
CCGCGAGACC
AGCCGGAAGG
TCCAGTCTAT
AATAGTTTGC
CTCGTCGTTT
GAGTTACATG
CCTCCGATCG
TATGGCAGCA
TTTCTGTGAC
CGGCGACCGA
ACATAGCAGA
GAAAACTCTC
ACTCGTGCAC
TGGGTGAGCA
CGACACGGAA
AGCATTTATC
TTAGAAAAAT
CACCTGACGT
AGGCGTATCA
AAACCTCTGA
CGGATGCCGG
GGGTGTCGGG
AGTGCACCAT

AAGCTCCCTC
TGTCCGCCTT
TGTAGGTATC
GCACGAACCC
GTCTTGAGTC
ACTGGTAACA
CTTGAAGTGG
TCTGCGCTCT
TGATCCGGCA
GCAGCAGATT
TTTCTACGGG
TTGGTCATGA
AAAATGAAGT
ACAGTTACCA
TTTCGTTCAT
ACGGGAGGGC
CACGCTCACC
GCCGAGCGCA
TAATTGTTGC
GCAACGTTGT
GGTATGGCTT
ATCCCCCATG
TTGTCAGAAG
CTGCATAATT
TGGTGAGTAC
GTTGCTCTTG
ACTTTAAAAG
AAGGATCTTA
CCAACTGATC
AAAACAGGAA
ATGTTGAATA
AGGGTTATTG
AAACAAATAG
CTAAGAAACC
CGAGGCCCTT
CACATGCAGC
GAGCAGACAA
GCTGGCTTAA
ATGCGGTGTG

GTGCGCTCTC
TCTCCCTTCG
TCAGTTCGGT
CCCGTTCAGC
CAACCCGGTA
GGATTAGCAG
TGGCCTAACT
GCTGAAGCCA
AACAAACCAC
ACGCGCAGAA
GTCTGACGCT
GATTATCAAA
TTTAAATCAA
ATGCTTAATC
CCATAGTTGC
TTACCATCTG
GGCTCCAGAT
GAAGTGGTCC
CGGGAAGCTA
TGCCATTGCT
CATTCAGCTC
TTGTGCAAAA
TAAGTTGGCC
CTCTTACTGT
TCAACCAAGT
CCCGGCGTCA
TGCTCATCAT
CCGCTGTTGA
TTCAGCATCT
GGCAAAATGC
CTCATACTCT
TCTCATGAGC
GGGTTCCGCG
ATTATTATCA
TCGTCTCGCG
TCCCGGAGAC
GCCCGTCAGG
CTATGCGGCA
AAATACCGCA

CTGTTCCGAC
GGAAGCGTGG
GTAGGTCGTT
CCGACCGCTG
AGACACGACT
AGCGAGGTAT
ACGGCTACAC
GTTACCTTCG
CGCTGGTAGC
AAAAAGGATC
CAGTGGAACG
AAGGATCTTC
TCTAAAGTAT
AGTGAGGCAC
CTGACTCCCC
GCCCCAGTGC
TTATCAGCAA
TGCAACTTTA
GAGTAAGTAG
ACAGGCATCG
CGGTTCCCAA
AAGCGGTTAG
GCAGTGTTAT
CATGCCATCC
CATTCTGAGA
ATACGGGATA
TGGAAAACGT
GATCCAGTTC
TTTACTTTCA
CGCAAAAAAG
TCCTTTTTCA
GGATACATAT
CACATTTCCC
TGACATTAAC
CGTTTCGGTG
GGTCACAGCT
GCGCGTCAGC
TCAGAGCAGA
CAGATGCGTA

CCTGCCGCTT
CGCTTTCTCA
CGCTCCAAGC
CGCCTTATCC
TATCGCCACT
GTAGGCGGTG
TAGAAGAACA
GAAAAAGAGT
GGTGGTTTTT
TCAAGAAGAT
AAAACTCACG
ACCTAGATCC
ATATGAGTAA
CTATCTCAGC
GTCGTGTAGA
TGCAATGATA
TAAACCAGCC
TCCGCCTCCA
TTCGCCAGTT
TGGTGTCACG
CGATCAAGGC
CTCCTTCGGT
CACTCATGGT
GTAAGATGCT
ATAGTGTATG
ATACCGCGCC
TCTTCGGGGC
GATGTAACCC
CCAGCGTTTC
GGAATAAGGG
ATATTATTGA
TTGAATGTAT
CGAAAAGTGC
CTATAAAAAT
ATGACGGTGA
TGTCTGTAAG
GGGTGTTGGC
TTGTACTGAG
AGGAGAAAAT

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# TB086
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Printed in USA.
Revised 1/03

2551
2601
2651
2701
2751
2801
2851
2901
2951
3001
3051
3101
3151

ACCGCATCAG
ATTTTTGTTA
ATCCCTTATA
AGTTTGGAAC
GGCGAAAAAC
TAATCAAGTT
TAAAGGGAGC
CGAGAAAGGA
AGTGTAGCGG
GCCGCTACAG
AGGGCGATCG
GATGTGCTGC
CGACGTTGTA

GAAATTGTAA
AATCAGCTCA
AATCAAAAGA
AAGAGTCCAC
CGTCTATCAG
TTTTGGGGTC
CCCCGATTTA
AGGGAAGAAA
TCACGCTGCG
GGCGCGTCCA
GTGCGGGCCT
AAGGCGATTA
AAACGACGGC

GCGTTAATAT
TTTTTTAACC
ATAGACCGAG
TATTAAAGAA
GGCGATGGCC
GAGGTGCCGT
GAGCTTGACG
GCGAAAGGAG
CGTAACCACC
TTCGCCATTC
CTTCGCTATT
AGTTGGGTAA
CAGTGAATTG

TTTGTTAAAA
AATAGGCCGA
ATAGGGTTGA
CGTGGACTCC
CACTACGTGA
AAAGCACTAA
GGGAAAGCCG
CGGGCGCTAG
ACACCCGCCG
AGGCTGCGCA
ACGCCAGCTG
CGCCAGGGTT
TAATACGACT

TTCGCGTTAA
AATCGGCAAA
GTGTTGTTCC
AACGTCAAAG
ACCATCACCC
ATCGGAACCC
GCGAACGTGG
GGCGCTGGCA
CGCTTAATGC
ACTGTTGGGA
GCGAAAGGGG
TTCCCAGTCA
CACTATA

VI. Related Products

Product
pGEM-3Z Vector(a)
pGEM-4Z 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
20g
20g
20g
20g
20g
20g
20g
20g
20g
20g
20g

Cat.#
P2151
P2161
P2261
P2241
P2351
P2251
P2371
P2391
P2411
P2421
P2541

Product
pSP64 Poly(A) Vector
pSP72 Vector(a)
pSP73 Vector(a)

Size
20g
20g
20g

Cat.#
P1241
P2191
P2221

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
2g
2g
2g
2g
2g
2g

Cat.#
Q5011
Q5021
Q5401
Q5391
Q5601
Q5421

All pGEM-Zf Vectors are provided with a glycerol stock of bacterial strain JM109.

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 1/03

Part# TB086
Page 7

Riboprobe in vitro Transcription Systems

Product
Riboprobe System - SP6(b)
Riboprobe System - T3(b)
Riboprobe System - T7(b)

Cat.#
P1420
P1430
P1440

VII. Reference
1. Yanish-Perron, C. et al. (1985) Improved M13 phage cloning vectors and host strains:
Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 10319.

(a)U.S. Pat. No. 4,766,072

has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted

(b)U.S. Pat. No. 5,552,302,

European Pat. No. 0 422 217, Australian Pat. No. 646803 and Japanese Pat. No. 3009458 have been issued to
Promega Corporation for the methods and compositions for production of human recombinant placental ribonuclease inhibitor.
19902002 Promega Corporation. All Rights Reserved.
pGEM and Riboprobe are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.
DH5 is a registered trademark of Life Technologies, Inc. DNASTAR is a registered trademark of DNASTAR, Inc. GenBank is a registered trademark of the U.S. Health and Human Services.
Promega Corporation
2800 Woods Hollow Road
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.

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

Part# TB086
Page 8

Printed in USA.
Revised 1/03