Promega P2261 pGEM-3Zf Vector Instruction Manual
- June 12, 2024
- Promega
Table of Contents
Promega P2261 pGEM-3Zf Vector Instruction Manual
All technical literature is available at:
www.promega.com/protocols/ Visit the web
site to verify that you are using the most current version of this Technical
Bulletin.
E-mail Promega Technical Services if you have questions on use of this
system: [email protected]
Description
The pGEM®-3Zf(–) Vector is a derivative of the pGEM®-3Z Vector. The plasmid serves as a standard cloning vector, and as a template for in vitro transcription.
The pGEM®-3Zf(–) Vector contains SP6 and T7 RNA polymerase promoters flanking the multiple cloning region within the α-peptide coding region of β-galactosidase (1). Insertional inactivation of the a-peptide allows recombinant clones to be directly identified by color screening on indicator plates.
The sequences of Promega vectors are available online at: www.promega.com/vectors/ and from the GenBank® database.
Product Components and Storage Conditions
| PRODUC T | SIZE | CAT. # |
|---|---|---|
| pGEM®-3Zf(–) Vector | 20µg | P2261 |
Storage Conditions: Store the pGEM®-3Zf(–) Vector at –30°C to –10°C.
pGEM®-3Zf(–) Vector Multiple Cloning Region and Circle Map
Figure 1. pGEM®-3Zf(–) Vector promoter and multiple cloning region sequence. The sequence shown corresponds to RNA synthesized by T7 RNA polymerase and is complementary to RNA synthesized by SP6 RNA polymerase.
Figure 2. pGEM®-3Zf(–) Vector circle map.
pGEM®-3Zf(–) Vector sequence reference points:
| T7 RNA Polymerase transcription initiation site | 1 |
|---|---|
| multiple cloning region | 5–61 |
| SP6 RNA polymerase promoter (–17 to +3) | 67–86 |
| SP6 RNA polymerase transcription initiation site | 69 |
| lac operon sequences | 94–323; 3018–3178 |
| lac Z start codon | 108 |
| lac operator | 128–144 |
| β-lactamase (Amp®) coding region | 1265–2125 |
| T7 RNA polymerase promoter (–17 to +3) | 3181–3 |
Specialized applications of the pGEM®-3Zf(–) Vector:
- Blue/white screening for recombinants.
- Transcription in vitro from dual-opposed promoters (For protocol information, please request the Riboprobe® in vitro Transcription Systems Technical Manual, #TM016.)
- Translation in vitro (For protocol information, please request the TNT® Quick Coupled Transcription/Translation System Technical Manual, #TM045.)
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 or [email protected]
Vector sequences are available in the GenBank® database (GenBank®/EMBL Accession Number X65307) and on the Internet at: www.promega.com/vectors/
Table 1. Restriction Enzymes That Cut the pGEM®-3Zf(–) Vector Between 1 and 5 Times.
| Enzyme | # of Sites | Location |
|---|---|---|
| AatII | 1 | 2260 |
| AccI | 1 | 39 |
| Acc65I | 1 | 17 |
| AcyI 2 | 2 | 1875, 2257 |
| AflIII | 1 | 445 |
| Alw26I | 4 | 1399, 2175, 2328, 2370 |
| Alw44I | 3 | 759, 2005, 2502 |
| AlwNI | 1 | 861 |
| AspHI | 5 | 15, 763, 1924, 2009, 2506 |
| AvaI | 1 | 21 |
| AvaII | 2 | 1476, 1698 |
| BamHI | 1 | 26 |
| BanI | 4 | 17, 189, 1286, 2751 |
| BanII | 2 | 15, 2721 |
| BbuI | 1 | 54 |
| BglI | 2 | 1458, 3030 |
| Bsa I | 1 | 1399 |
| BsaAI | 1 | 2792 |
| BsaHI | 2 | 1875, 2257 |
| BsaJI | 5 | 21, 22, 184, 605, 3133 |
| BsaOI | 5 | 361, 785, 1708, 1857, 3058 |
| BspHI | 3 | 1165, 2173, 2278 |
| BspMI | 1 | 51 |
| BssSI | 3 | 618, 2002, 2309 |
| BstOI | 5 | 185, 473, 594, 607, 3134 |
| Cfr10I | 2 | 1418, 2687 |
| DraI | 3 | 1204, 1223, 1915 |
| DraII | 1 | 2314 |
| DraIII | 1 | 2795 |
| DrdI | 3 | 553, 2422, 2839 |
| EaeI | 3 | 284, 1726, 3167 |
| EarI | 3 | 329, 2133, 3075 |
| EclHKI | 1 | 1338 |
| EcoICRI | 1 | 13 |
| EcoRI | 1 | 5 |
| FokI | 5 | 1304, 1485, 1772, 2415, 3113 |
| FspI | 2 | 1560, 3037 |
| HaeII | 4 | 323, 693, 2637, 2645 |
| HgaI | 5 | 556, 1134, 1864, 2422, 2570 |
| HincII | 1 | 40 |
| HindII | 1 | 40 |
| HindIII | 1 | 56 |
| Hsp92I | 2 | 1875, 2257 |
| KpnI | 1 | 21 |
| MaeI | 5 | 33, 940, 1193, 1528, 2639 |
| NaeI | 1 | 2689 |
| NdeI | 1 | 2509 |
| NgoMIV | 1 | 2687 |
| NspI | 3 | 54, 449, 2366 |
| PspAI | 1 | 21 |
| PstI | 1 | 48 |
| PvuI | 2 | 1708, 3058 |
| PvuII | 2 | 269, 3087 |
| RsaI | 3 | 19, 1818, 2494 |
| SacI | 1 | 15 |
| SalI | 1 | 38 |
| ScaI | 1 | 1818 |
| SinI | 2 | 1476, 1698 |
| Smal | 1 | 23 |
| SphI | 1 | 54 |
| Sse8387I | 1 | 48 |
| SspI | 2 | 2142, 3000 |
| TaqI | 5 | 9, 39, 545, 1989, 2757 |
| TfiI | 2 | 280, 420 |
| VspI | 3 | 216, 275, 1510 |
| XbaI | 1 | 32 |
| XmaI | 1 | 21 |
| XmnI | 1 | 1937 |
Table 2. Restriction Enzymes That Do Not Cut the pGEM®-3Zf(–) Vector.
| AccIII | BlpI | Bsu36I | FseI | PflMI | SnaBI |
|---|---|---|---|---|---|
| AccB7I | Bpu1102I | ClaI | HpaI | PinAI | SpeI |
| AflII | BsaBI | CspI | I-PpoI | PmeI | SplI |
| AgeI | BsaMI | Csp45I | KasI | PmlI | SrfI |
| ApaI | BsmI | DsaI | MluI | Ppu10I | StuI |
| AscI | Bsp120I | EagI | NarI | PpuMI | StyI |
| AvrII | BsrGI | Eco47III | NcoI | PshAI | SwaI |
| BalI | BssHII | Eco52I | NheI | Psp5II | Tth111I |
| BbeI | Bst1107I | Eco72I | NotI | RsrII | XcmI |
| BbrPI | Bst98I | Eco81I | NruI | SacII | XhoI |
| Bbsl | BstEll | EcoNl | Nsil | Sfil | |
| Bcll | BstXI | EcoRV | PacI | Sgfl | |
| Bgll | BstZl | Ehel | PaeR7l | SgrAl |
Table 3. Restriction Enzymes That Cut the pGEM®-3Zf(–) Vector 6 or More Times.
| AciI | CfoI | HpaII | MseI | Sau3AI |
|---|---|---|---|---|
| AluI | DdeI | HphI | MspI | Sau96I |
| BbvI | DpnI | Hsp92II | MspA1I | ScrFI |
| Bsp1286I | DpnII | MaeII | NciI | SfaNI |
| BsrI | Fnu4HI | MaeIII | NdeII | Tru9I |
| BsrSI | HaeIII | MboI | NlaIII | XhoII |
| Bst71I | HhaI | MboII | NlaIV | |
| BstUI | HinfI | MnlI | PleI |
Related Products
pGEM® Vectors
| Product | Size | Cat.# |
|---|---|---|
| pGEM®-3Z Vector | 20µg | P2151 |
| pGEM®-4Z Vector | 20µg | P2161 |
| pGEM®-3Zf(+) Vector | 20µg | P2271 |
| pGEM®-5Zf(+) Vector | 20µg | P2241 |
| pGEM®-7Zf(+) Vector | 20µg | P2251 |
For Research Use Only. Not for Use in Diagnostic Procedures.
Other Vectors
| Product | Size | Cat.# |
|---|---|---|
| pSP64 Poly(A) Vector | 20µg | P1241 |
| pSP72 Vector | 20µg | P2191 |
| pSP73 Vector | 20µg | P2221 |
For Research Use Only. Not for Use in Diagnostic Procedures.
Sequencing Primer
| Product | Size | Cat.# |
|---|---|---|
| SP6 Promoter Primer | 2µg | Q5011 |
For Research Use Only. Not for Use in Diagnostic Procedures.
In Vitro Transcription Systems
| Product | Cat.# |
|---|---|
| Riboprobe® System—SP6 | P1420 |
| Riboprobe® System—T3 | P1430 |
| Riboprobe® System—T7 | P1440 |
| RiboMAX™ Large Scale RNA Production Systems, SP6 | P1280 |
| RiboMAX™ Large Scale RNA Production Systems T7 | P1300 |
| T7 RiboMAX™ Express Large Scale RNA Production System | P1320 |
For Research Use Only. Not for Use in Diagnostic Procedures.
TNT® Quick Coupled Transcription/Translation Systems
| Product | Cat.# |
|---|---|
| TNT® T7 Quick Coupled Transcription/Translation System | L1170 |
| TNT® T7 Quick Coupled Transcription/Translation System, Trial Size | L1171 |
| TNT® SP6 Quick Coupled Transcription/Translation System | L2080 |
| TNT® SP6 Quick Coupled Transcription/Translation System, Trial Size | L2081 |
Summary of Changes
The following changes were made to the 2/23 revision of this document:
- Removed JM109 cells due to discontinuation.
- Updated Related Products.
- Updated font and cover image.
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