pmono-hygro-gfp - invivogen.com · nhei (3741) hpai (3881) mfei (3890) paci (3985) ... sni l...
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pMONO-hygro-GFPA GFP-expression plasmid selectable with Hygromycin
Catalog # pmonoh-gfp
For research use onlyVersion # 04A29-MT
PRODUCT INFORMATIONContent:- 20 µg of pMONO-hygro-gfp 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 should be resuspended upon receipt and stored at - 20˚C.Lyophilized DNA is stable 12 months at -20˚C. Resuspended DNA is stablemore than one year 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.
GENERAL PRODUCT USEpMONO plasmids are specifically designed for strong and constitutiveexpression of a gene of interest in a wide variety of cell lines. They allowthe selection of stable transfectants and offer a choice of selectablemarkers. pMONO plasmids contain a unique transcription unit that drivesthe expression of the gene of interest and the selectable marker through aninternal ribosome entry site (IRES). This dual gene expression systemensures that stable clones express the gene of interest.pMONO-GFP plasmids feature a new allele of the GFP gene called LGFP.They can be used as control vectors or for cloning of an open readingframe, as the LGFP gene is flanked by two unique restriction sites: BspHI at the 5’ end that encompasses the Start codon, and Avr II at the 3’end.
PLASMID FEATURES• SV40/FerH/mEF1a: pMONO plasmids feature a composite ferritinpromoter that confers strong and constitutive expression in a widerange of mammalian cells. The promoter is composed of the ferritinheavy chain (FerH) core promoter1 fused at its 5’ end to the SV40enhancer, and at its 3’ end to the intron-containing 5’UTR of the mouseelongation factor 1 alpha gene. This composite promoter yields similarlevels of expression as the CMV promoter in all cell lines tested.• LGFP: This red-shifted variant of the jellyfish GFP gene encodes agreen fluorescent protein that absorbs blue light (major peak at 480 nm)and emits green light (major peak at 505 nm).• FMDV IRES: The internal ribosome entry site of the Foot and MouthDisease Virus enables the translation of two open reading frames fromone mRNA with high levels of expression2.• Hygro: Resistance to Hygromycin B is conferred by the hph gene fromE. coli which encodes a phosphotransferase. In mammalian cells, the hphgene is transcribed from the composite ferritin promoter as a polycistronicmRNA and translated through the FMDV IRES. In E. coli, hph istranscribed from the bacterial EM7 promoter.
• EM7 is a bacterial promoter that enables the constitutive expression ofthe antibiotic resistance gene in E. coli.• SV40 pAn: the Simian Virus 40 late polyadenylation signal enablesefficient cleavage and polyadenylation reactions resulting in high levelsof steady-state mRNA3.• ori: a minimal E. coli origin of replication to limit vector size, but withthe same activity as the longer Ori.
METHODSPlasmid resuspension:Quickly spin the tube containing the lyophilized plasmid to pellet theDNA. To obtain a plasmid solution at 1 µg/µl, resuspend the DNA in 20µl 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 solidmedia for bacterial culture by using only a microwave. Fast-Media® is aTB (liquid) or LB (solid) based medium that already contains theantibiotic.F a s t - M e d i a® Hygro can be ordered separately (#fas-hg-l (liquid), #fas-hg-s( s o l i d ) ) .
Method:1- Pour the contents of a Fast-Media® pouch into a clean borosilicate glassbottle or flask.2- Add 200 ml of distilled water to the flask3- Heat in a microwave on MEDIUM power setting (about 400Watts),until bubbles start appearing (approximately 3 minutes). Do not heat aclosed container. Do not autoclave Fast-Media®.4- Swirl gently to mix the preparation. Be careful, the bottle and mediaare hot, use heatproof pads or gloves and care when handling.5- Reheat the media for 30 seconds and gently swirl again. Repeat asnecessary to completely dissolve the powder into solution. But be carefulto avoid overboiling and volume loss.6- Let agar medium cool to 45˚C before pouring plates. Let liquid mediacool to 37˚C before seeding bacteria. Note: Do not reheat solidified Fast-Media® as the antibiotic will bepermanently destroyed by the procedure.
References:1. Eisenstein RS. and Munro HN. 1990. Translational regulation of ferritin synthesis by iron.Enzyme 44(1-4):42-582. Ramesh N et al. 1996. High-titer bicistronic retroviral vectors employing foot-and-mouthdisease virus internal ribosome entry site. Nucleic Acids Res. 24(14):2697-7003. Carswell S. & Alwine JC. 1989. Efficiency of utilization of the simian virus 40 latepolyadenylation site: effects of upstream sequences. Mol. Cell Biol. 10: 4248-4258
TECHNICAL SUPPORTToll free (US): 888-457-5873Outside US: (+1) 858-457-5873E-mail: info@invivogen.comWebsite: www.invivogen.com
3950 Sorrento Valley Blvd. Suite ASan Diego, CA 92121 - USA
125
pMONO-hygro-GFP(4728 bp)
SV40 enh
FMDV IRES
ori
SV40 pAn
EM7
FerH prom
mEF1 5'UTR
LGFP
Hygro
SdaI (6)
PvuII (69)SpeI (243)
NotI (276)HindIII (428)
SacII (472)
SmaI (1049)
SpeI (1278)
EcoNI (1391)AgeI (1428)
BspHI (1440)
HpaI (1801)
AvrII (2160)XbaI (2225)Eco47III (2551)
HindIII (2564)AseI (2651)
BbsI (3460)
NheI (3741)
HpaI (3881)MfeI (3890)
PacI (3985)
ApaLI (4399)
PacI (4725)
CCTGCAGGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCTGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAA
AGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAG
TATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCACTAGTTCCGCCAGAGCGCGCGAGGGCCTCCAGCGGCCGCCCCTCCCCCACAGCAGGG
GCGGGGTCCCGCGCCCACCGGAAGGAGCGGGCTCGGGGCGGGCGGCGCTGATTGGCCGGGGCGGGCCTGACGCCGACGCGGCTATAAGAGACCACAAGCG
ACCCGCAGGGCCAGACGTTCTTCGCCGAAGCTTGCCGTCAGAACGCAGGTGAGGGGCGGGTGTGGCTTCCGCGGGCCGCCGAGCTGGAGGTCCTGCTCCG
AGCGGGCCGGGCCCCGCTGTCGTCGGCGGGGATTAGCTGCGAGCATTCCCGCTTCGAGTTGCGGGCGGCGCGGGAGGCAGAGTGCGAGGCCTAGCGGCAA
CCCCGTAGCCTCGCCTCGTGTCCGGCTTGAGGCCTAGCGTGGTGTCCGCGCCGCCGCCGCGTGCTACTCCGGCCGCACTCTGGTCTTTTTTTTTTTTGTT
GTTGTTGCCCTGCTGCCTTCGATTGCCGTTCAGCAATAGGGGCTAACAAAGGGAGGGTGCGGGGCTTGCTCGCCCGGAGCCCGGAGAGGTCATGGTTGGG
GAGGAATGGAGGGACAGGAGTGGCGGCTGGGGCCCGCCCGCCTTCGGAGCACATGTCCGACGCCACCTGGATGGGGCGAGGCCTGGGGTTTTTCCCGAAG
CAACCAGGCTGGGGTTAGCGTGCCGAGGCCATGTGGCCCCAGCACCCGGCACGATCTGGCTTGGCGGCGCCGCGTTGCCCTGCCTCCCTAACTAGGGTGA
GGCCATCCCGTCCGGCACCAGTTGCGTGCGTGGAAAGATGGCCGCTCCCGGGCCCTGTTGCAAGGAGCTCAAAATGGAGGACGCGGCAGCCCGGTGGAGC
GGGCGGGTGAGTCACCCACACAAAGGAAGAGGGCCTGGTCCCTCACCGGCTGCTGCTTCCTGTGACCCCGTGGTCCTATCGGCCGCAATAGTCACCTCGG
GCTTTTGAGCACGGCTAGTCGCGGCGGGGGGAGGGGATGTAATGGCGTTGGAGTTTGTTCACATTTGGTGGGTGGAGACTAGTCAGGCCAGCCTGGCGCT
GGAAGTCATTTTTGGAATTTGTCCCCTTGAGTTTTGAGCGGAGCTAATTCTCGGGCTTCTTAGCGGTTCAAAGGTATCTTTTAAACCCTTTTTTAGGTGT
TGTGAAAACCACCGCTAATTCAAAGCAACCGGTCGCACGTCATGAGCAAGGGAGAAGAACTCTTTACTGGTGTTGTCCCAATTCTGGTTGAGCTGGATGG
TGATGTGAATGGCCACAAATTCTCTGTGTCTGGTGAAGGTGAAGGAGATGCAACTTATGGAAAGCTGACTCTGAAGTTCATTTGTACAACAGGAAAGCTG
CCAGTGCCTTGGCCAACTCTGGTGACCACCCTGACTTATGGTGTTCAATGTTTCAGCAGGTACCCTGACCACATGAAGCAGCATGACTTCTTTAAATCTG
CAATGCCAGAAGGTTATGTTCAGGAGAGGACAATCTTCTTTAAGGATGATGGAAATTATAAGACAAGGGCAGAAGTGAAGTTTGAAGGTGATACACTGGT
TAACAGAATTGAGCTGAAAGGCATTGATTTTAAGGAAGATGGAAACATTCTGGGTCACAAGCTGGAGTACAACTATAATTCTCACAATGTTTACATTATG
GCAGATAAGCAGAAGAATGGAATTAAGGTTAATTTCAAGATTAGACACAACATTGAGGATGGATCTGTCCAACTGGCAGACCATTACCAGCAGAACACCC
CTATTGGTGATGGCCCAGTTCTCCTCCCAGATAATCACTATCTCCGCACTCAATCTGCTCTGTCCAAAGACCCTAATGAGAAAAGAGACCACATGGTCCT
CCTGGAGTTTGTGACAGCAGCAGGAATTACTCTGGGAATGGATGAGCTGTACAAGTAAACCTAGGAGCAGGTTTCCCCAATGACACAAAACGTGCAACTT
GAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTG
CTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCC
CAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGG
TAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCA
CCTTTCCTTTGCAATTACTGACCCTATGAATACAACTGACTGTTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGG
AGGGCCACCATGAAGAAACCTGAACTGACAGCAACTTCTGTTGAGAAGTTTCTCATTGAAAAATTTGATTCTGTTTCTGATCTCATGCAGCTGTCTGAAG
GTGAAGAAAGCAGAGCCTTTTCTTTTGATGTTGGAGGAAGAGGTTATGTTCTGAGGGTCAATTCTTGTGCTGATGGTTTTTACAAAGACAGATATGTTTA
CAGACACTTTGCCTCTGCTGCTCTGCCAATTCCAGAAGTTCTGGACATTGGAGAATTTTCTGAATCTCTCACCTACTGCATCAGCAGAAGAGCACAAGGA
GTCACTCTCCAGGATCTCCCTGAAACTGAGCTGCCAGCTGTTCTGCAACCTGTTGCTGAAGCAATGGATGCCATTGCAGCAGCTGATCTGAGCCAAACCT
CTGGATTTGGTCCTTTTGGTCCCCAAGGCATTGGTCAGTACACCACTTGGAGGGATTTCATTTGTGCCATTGCTGATCCTCATGTCTATCACTGGCAGAC
TGTGATGGATGACACAGTTTCTGCTTCTGTTGCTCAGGCACTGGATGAACTCATGCTGTGGGCAGAAGATTGTCCTGAAGTCAGACACCTGGTCCATGCT
Me tLysLysP roGl uLeuThr Al aThr Ser Va l Gl uLysPheLeu I l eGl uLysPheAspSer Va l SerAspLeuMe tGl nLeuSer Gl uG
l yGl uGl uSer A rgAl aPheSer PheAspVa l Gl yGl yA rgGl yTyrVa l LeuArgVa l AsnSer CysAl aAspGl yPheTyrLysAspArgTyrVa l Ty
r A rgHi s PheAl aSer Al aAl aLeuP ro I l eP roGl uVa l LeuAsp I l eGl yGl uPheSer Gl uSer LeuThr Ty rCys I l eSer A rgArgAl aGl nGl y
Va l Thr LeuGl nAspLeuP roGl uThr Gl uLeuP roAl aVa l LeuGl nP roVa l A l aGl uAl aMe tAspAl a I l eAl aAl aAl aAspLeuSer Gl nThr S
er Gl yPheGl y P roPheGl y P roGl nGl y I l eGl yGl nTy rThr Thr T rpArgAspPhe I l eCysAl a I l eAl aAspP roHi sVa l Ty rHi sT rpGl nTh
r Va l Me tAspAspThr Va l Ser Al aSer Va l A l aGl nAl aLeuAspGl uLeuMe tLeuT rpAl aGl uAspCysP roGl uVa l A rgHi s LeuVa l Hi sAl a
aTyrMe tLeuArg I l eGl yLeuAspGl nLeuTyrGl nSer LeuVa l AspGl yAsnPheAspAspAl aAl aT rpAl aGl nGl yA rgCysAspAl a I l eVa l
A rgSer Gl yA l aGl yThr Va l Gl yA rgThr Gl n I l eAl aArgArgSer Al aAl aVa l T rpThrAspGl yCysVa l Gl uVa l LeuAl aAspSer Gl yAsnA
r gArgP roSer Thr A rgP roArgAl aLysGl u•••
Me tSer LysGl yGl uGl uLeuPheThr Gl yVa l Va l P ro I l eLeuVa l Gl uLeuAspGl
yAspVa l AsnGl yHi s LysPheSer Va l Ser Gl yGl uGl yGl uGl yAspAl aThr Ty rGl yLysLeuThr LeuLysPhe I l eCysThr Thr Gl yLysLeu
P roVa l P roT rpP roThr LeuVa l Thr Thr LeuThr Ty rGl yVa l Gl nCysPheSer A rgTyrP roAspHi sMe tLysGl nHi sAspPhePheLysSer A
l aMe tP roGl uGl yTyrVa l Gl nGl uArgThr I l ePhePheLysAspAspGl yAsnTyrLysThr A rgAl aGl uVa l LysPheGl uGl yAspThr LeuVa
l AsnArg I l eGl uLeuLysGl y I l eAspPheLysGl uAspGl yAsn I l eLeuGl yHi s LysLeuGl uTyrAsnTyrAsnSer Hi sAsnVa l Ty r I l eMe t
A l aAspLysGl nLysAsnGl y I l eLysVa l AsnPheLys I l eArgHi sAsn I l eGl uAspGl ySer Va l Gl nLeuAl aAspHi sTy rGl nGl nAsnThr P
r o I l eGl yAspGl y P roVa l LeuLeuP roAspAsnHi sTy rLeuArgThr Gl nSer Al aLeuSer LysAspP roAsnGl uLysArgAspHi sMe tVa l Le
uLeuGl uPheVa l Thr Al aAl aGl y I l eThr LeuGl yMe tAspGl uLeuTyrLys•••
SdaI (6) PvuII (69)
SpeI (243) NotI (276)
HindIII (428) SacII (472)
SmaI (1049)
SpeI (1278)
EcoNI (1391)
AgeI (1428) BspHI (1440)
HpaI (1801)
AvrII (2160)
XbaI (2225)
Eco47III (2551) HindIII (2564)
AseI (2651)
1
101
201
301
401
501
601
701
801
901
1001
1101
1201
1301
1401
1501
1601
1701
1801
1901
2001
2101
2201
2301
2401
2501
2601
2701
2801
2901
3001
3101
3201
1
31
64
98
131
164
264
298
331
1
20
54
87
120
154
187
220
GATTTTGGAAGCAACAATGTTCTGACAGACAATGGCAGAATCACTGCAGTCATTGACTGGTCTGAAGCCATGTTTGGAGATTCTCAATATGAGGTTGCCA
ACATTTTTTTTTGGAGACCTTGGCTGGCTTGCATGGAACAACAAACAAGATATTTTGAAAGAAGACACCCAGAACTGGCTGGTTCCCCCAGACTGAGAGC
CTACATGCTCAGAATTGGCCTGGACCAACTGTATCAATCTCTGGTTGATGGAAACTTTGATGATGCTGCTTGGGCACAAGGAAGATGTGATGCCATTGTG
AGGTCTGGTGCTGGAACTGTTGGAAGAACTCAAATTGCAAGAAGGTCTGCTGCTGTTTGGACTGATGGATGTGTTGAAGTTCTGGCTGACTCTGGAAACA
GGAGACCCTCCACAAGACCCAGAGCCAAGGAATGAATCCTGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATG
CAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC
ATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAAATGTTAATTAACTAGCCATGACC
AAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT
GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA
GATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA
GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT
GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA
CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC
CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT
GGCCTTTTGCTCACATGTTCTTAATTAA
Me tLysLysP roGl uLeuThr Al aThr Ser Va l Gl uLysPheLeu I l eGl uLysPheAspSer Va l SerAspLeuMe tGl nLeuSer Gl uG
l yGl uGl uSer A rgAl aPheSer PheAspVa l Gl yGl yA rgGl yTyrVa l LeuArgVa l AsnSer CysAl aAspGl yPheTyrLysAspArgTyrVa l Ty
AspPheGl ySerAsnAsnVa l LeuThrAspAsnGl yA rg I l eThr Al aVa l I l eAspT rpSer Gl uAl aMe tPheGl yAspSer Gl nTy rGl uVa l A l aA
sn I l ePhePheT rpArgP roT rpLeuAl aCysMe tGl uGl nGl nThr A rgTyrPheGl uArgArgHi s P roGl uLeuAl aGl ySer P roArgLeuArgAl
aTyrMe tLeuArg I l eGl yLeuAspGl nLeuTyrGl nSer LeuVa l AspGl yAsnPheAspAspAl aAl aT rpAl aGl nGl yA rgCysAspAl a I l eVa l
A rgSer Gl yA l aGl yThr Va l Gl yA rgThr Gl n I l eAl aArgArgSer Al aAl aVa l T rpThrAspGl yCysVa l Gl uVa l LeuAl aAspSer Gl yAsnA
r gArgP roSer Thr A rgP roArgAl aLysGl u•••
BbsI (3460)
NheI (3741)
HpaI (3881) MfeI (3890)
PacI (3985)
ApaLI (4399)
PacI (4725)
3301
3401
3501
3601
3701
3801
3901
4001
4101
4201
4301
4401
4501
4601
4701
1
31
198
231
264
298
331
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