pSELECT-hygro-mcs

pSELECT-hygro-mcs
Dual expression cassette plasmid for the expression of one gene of interest
Catalog # pseth-mcs
For research use only
Version # 04L20-SV
PRODUCT INFORMATION
Second expression cassette
Contents:
- 20 µg of pSELECT-hygro-mcs plasmid provided as lyophilized DNA
- 4 pouches of E. coli Fast-Media® Hygro (2 TB and 2 Agar)
Storage and Stability:
Product is shipped at room temperature. Lyophilized DNA is stable 1 year
when stored at -20˚C. Resuspended DNA is stable 12 months when stored
at -20˚C. Avoid repeated freeze-thaw cycles.
Store E. coli Fast-Media® Hygro at room temperature. Fast-Media®
pouches are stable 18 months when stored properly.
Quality control:
Plasmid construct has been confirmed by restriction analysis and sequencing.
Plasmid DNA was purified by ion exchange chromatography and lyophilized.
• CMV enh/prom: The human cytomegalovirus immediate-early gene 1
promoter/enhancer was originally isolated from the Towne strain and was
found to be stronger than any other viral promoters.
• EM7 is a bacterial promoter that enables the constitutive expression of
the antibiotic resistance gene in E. coli.
• Hygro: Resistance to Hygromycin B is conferred by the hph gene from
E. coli which encodes a phosphotransferase. The hph gene is driven by the
CMV enhancer/promoter in tandem with the bacterial EM7 promoter
allowing selection in both mammalian cells and E. coli.
• ßGlo pAn: The human beta-globin 3’UTR and polyadenylation
sequence allows efficient arrest of the transgene transcription4.
GENERAL PRODUCT USE
Plasmid resuspension:
Quickly spin the tube containing the lyophilized plasmid to pellet the
DNA. To obtain a plasmid solution at 1 µg/µl, resuspend the DNA in 20
µl of sterile H2O. Store resuspended plasmid at -20°C.
Selection of bacteria with E. coli Fast-Media®
Fast-Media® is a fast and convenient way to prepare liquid and solid
media for bacterial culture by using only a microwave. Fast-Media® is a
TB (liquid) or LB (solid) based medium that already contains the
antibiotic.
Fast-Media® Hygro can be ordered separately [#fas-hg-l (liquid), #fas-hg-s
(solid)].
pSELECT plasmids are specifically designed for strong and constitutive
expression of a gene of interest in a wide variety of cell lines. They allow
the selection of stable transfectants and offer a variety of selectable
markers. pSELECT plasmids contain two expression cassettes: the first
drives the expression of the gene of interest and the second drives the
expression of a large choice of dominant selectable markers for both
E. coli and mammalian cells. They are both terminating with a strong
polyadenylation signal (polyA) that separates the two expression cassettes
thus preventing any transcription interference. The late SV40 polyA
terminates the transcription of the gene of interest while the human ß-globin
polyA terminates the transcription of the selectable marker.
Note: The use of the late SV40 polyA allows you to silence your gene of
i n t e rest by using the ready-made psiRNA-SV40pA (#psirna3gz21sv40pa), a plasmid expressing a short hairpin siRNA targeting the late
SV40 polyA.
PLASMID FEATURES
First expression cassette
• hEF1-HTLV prom is a composite promoter comprising the Elongation
Factor-1α (EF-1α) core promoter1 and the R segment and part of the U5
sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1
Long Terminal Repeat2. The EF-1α promoter exhibits a strong activity
and yields long lasting expression of a transgene in vivo. The R-U5’ has
been coupled to the EF-1α core promoter to enhance stability of RNA.
• MCS: The multiple cloning site contains the following restriction sites:
5’ - Sal I, SgrA I, BamH I, Eco47 III, Nco I, Nhe I - 3’
Each restriction site is compatible with many other enzymes, increasing
the cloning options.
• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables
efficient cleavage and polyadenylation reactions resulting in high levels
of steady-state mRNA3.
• Ori: a minimal E. coli origin of replication to limit vector size, but with
the same activity as the longer Ori.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
E-mail: [email protected]
Website: www.invivogen.com
METHODS
Method:
1- Pour the contents of a Fast-Media® pouch into a clean borosilicate glass
bottle or flask.
2- Add 200 ml of distilled water to the flask
3- Heat in a microwave on MEDIUM power setting (about 400Watts),
until bubbles start appearing (approximately 3 minutes). Do not heat a
closed container. Do not autoclave Fast-Media®.
4- Swirl gently to mix the preparation. Be careful, the bottle and media
are hot, use heatproof pads or gloves and care when handling.
5- Reheat the media for 30 seconds and gently swirl again. Repeat as
necessary to completely dissolve the powder into solution. But be careful
to avoid overboiling and volume loss.
6- Let agar medium cool to 45˚C before pouring plates. Let liquid media
cool to 37˚C before seeding bacteria.
Note: Do not reheat solidified Fast-Media® as the antibiotic will be
permanently destroyed by the procedure.
References:
1. Kim, D.W. et al. (1990). Gene 2: 217-223.
2. Takebe, Y. et al. (1988). Mol. Cell Biol. 1: 466-472.
3. Carswell, S., and Alwine, J.C. (1989). Mol. Cell Biol. 10: 4248-4258.
4. Yu J & Russell JE. (2001). Mol Cell Biol, 21(17):5879-88.
3950 Sorrento Valley Blvd. Suite A
San Diego, CA 92121 - USA
NotI (2)
SwaI (3815)
PacI (3807)
SgfI (176)
PvuII (407)
HindIII (411)
Bsu36I (458)
NgoMI (607)
Ori
hEF1/HTLV prom
BspLU11I (3073)
PacI (3067)
MCS
pSELECT-hygro-mcs
CMV enh/prom
SV40 pAn
SgrAI (718)
SalI (726)
BamHI (732)
Eco47III (740)
NcoI (750)
NheI (756)
HpaI (896)
(3821 bp)
ßGlo pAn
EM7
SwaI (1246)
AseI (2490)
Hygro
BspHI (2431)
SgfI (2071)
100
NotI (2)
1 GCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACA
SgfI (176)
101 AAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCA
201 GAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATG
301 TCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG
HindIII (411)
PvuII (407)
Bsu36I (458)
401 AACACAGCTGAAGCTTCGAG GGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCT
501 CCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA
NgoMI (607)
601 GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACC
SalI (726)
Eco47III (740)
NheI (756)
SgrAI (718)
BamHI (732)
NcoI (750)
701 GGCGCCTACCTGAGATCACCGGCGTGTCGACGGATCCAGCGCTCTGCAGCCATGGGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAA
HpaI (896)
801 ACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACA
901 ACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAATTCTAAAA
1001 TACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGC
1101 ATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCA
SwaI (1246)
1201 CTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGC
1301 TCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTAGCGAA
1401 TTCTCGACTCATTCCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTC
342 •••G l uLysA l a A r gP r oAr gTh r Se r P r oAr gAr gAs nG l ySe r A s pA l a LeuVa l G l uVa l Cys G l yAs pTh r T r pVa l A l aA l a Se r A r
1501 TGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAAC
311 gAr gA l a I l eG l nTh r A r gG l yVa l Th r G l yA l a G l ySe r A r gVa l I l eA l aAs pCysAr gG l yG l nA l a T r pA l aA l aAs pAs pPh eAs nG l yAs pVa l
1601 CAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGCCGCGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTC
278 LeuSe r G l nT y r LeuG l nAs pLeuG l y I l eAr gLeuMe t T y r A l a A r gLeuAr gP r oSe r G l yA l a LeuG l uP r oHi s A r gAr gG l uPh eT y r A r gTh r G
1701 TGCTGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTG
244 l nG l nG l uMe t CysA l a LeuT r pP r oAr gT r pPh ePh e I l eAs nA l a Va l G l uT y r G l nSe r A s pG l yPh eMe tA l a G l uSe r T r pAs p I l eVa l A l a Th
1801 TTATGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATC
211 r I l eAr gG l yAs nAs pTh r LeuVa l As nAs nSe r G l yPh eAs pA l a Hi s Va l LeuHi s A r gVa l G l uP r oCysAs pG l uA l a T r pLeuMe t LeuG l uAs p
1901 GAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATGTA
178 LeuA l a G l nA l a Va l Se r A l a Se r Va l Th r A s pAs pMe t Va l Th r G l nT r pHi s T y r Va l Hi s P r oAs pA l a I l eA l a Cys I l ePh eAs pAr gT r pTh r T
SgfI (2071)
2001 GTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCATGAGCTCCGCGACGGGTTGCA
144 hr T y r G l nG l y I l eG l yG l nP r oG l yPh eP r oG l yPh eG l ySe r Th r G l nSe r LeuAs pA l aA l aA l a I l eA l aAs pMe t LeuG l uA l a Va l P r oG l nLe
2101 GAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTGAATTCCCCAATGTC
111 uVa l A l a P r oLeuG l uTh r G l uP r oLeuAs pG l nLeuTh r Va l G l yG l nA l a A r gAr gSe r I l eCys T y r Th r LeuSe r G l uSe r Ph eG l uG l y I l eAs p
2201 AAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATAT
7 8 LeuVa l G l uP r o I l eP r oLeuA l aA l a Se r A l a Ph eHi s A r gT y r Va l T y r A r gAs pLy s T y r Ph eG l yAs pA l a Cys Se r A s nVa l A r gLeuVa l T y r G
2301 CCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCT
4 4 l yA r gG l yG l yVa l As pPh eSe r Ph eA l a A r gSe r G l uG l uG l yG l uSe r LeuG l nMe t LeuAs pSe r Va l Se r A s pPh eLy s G l u I l eLeuPh eLy s G l
BspHI (2431)
AseI (2490)
2401 CGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATGATGGCCCTCCTATAGTGAGTCGTATTATACTATGCCGATATACTATGCCGATGATTAATTGTCAA
1 1 uVa l Se r Th r A l a Th r LeuG l uP r oLy s Ly s Me t
2501 AACAGCGTGGATGGCGTCTCCAGCTTATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAA
2601 TGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTACTAGTCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGT
2701 GAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCATCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGT
2801 CCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGC
2901 CAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGG
PacI (3067)
BspLU11I (3073)
3001 CGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCTGCAGGTT AATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC
3101 CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC
3201 CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC
3301 TCCCTTCGGGAAGCGTGGCGCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC
3401 CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG
3501 ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGC
3601 TGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC
3701 GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCT
PacI (3807) SwaI (3815)
3801 AGTTAATTAACATTTAAATC A