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