pORF9-mIL-15
An expression vector containing the murine Interleukin 15 open reading frame
PRODUCT INFORMATION • mIL-15 Gene Content:
Murine IL-15 gene (intronless ORF) from the ATG to the stop codon.
- 1 disk of lyophilized GT100 E. coli bacteria transformed by pORF9-mIL-15.
- GT100 genotype is: F-, mcrA , Δ(mrr-hsdRMS-mcrBC) , Ø80lacZ∆M15 ,• SV40 pAn: The Simian Virus 40 late polyadenylation signal enables eff i c i e n t
cleavage and polyadenylation reactions resulting in high levels of steady-state
- 4 pouches of E. coli FastMedia™ Amp.
mRNA. The efficiency of this signal was first described by Carswell et al.3
Storage and stability: - Products are shipped at room temperature. References
- Transformed bacteria should be stored at -20°C and are stable up to 1 year.
1- Kim et al (1990). Gene 2: 217-223.
- Store E. coli FastMedia™ Amp at room temperature. FastMedia™ is stable
2- Takebe et al (1988). Mol. Cell Biol. 1: 466-472.
3- Carswell et al(1989). Mol. Cell Biol. 10: 4248-4258. Quality control: - Plasmid construct has been confirmed by restriction analysis and sequencing.
- Bacteria have been lyophilized, and their viability upon resuspension has
Growth of pORF-transformed bacteria: Use sterile conditions to do the following: 1- Resuspend the lyophilized E. coli by adding 1 ml of LB medium in the GENERAL PRODUCT USE
tube containing the disk. Let sit for 5 minutes. Mix gently by inverting the
pORF is a ready-made expression vector containing a gene of interest.
tube several times. 2- Streak bacteria taken from this suspension on an ampicillin LB agar plate
prepared with the E. coli FastMedia™ Amp agar provided (see below). Obtaining a gene to subclone into another vector. Two unique restriction
3- Place the plate in an incubator at 37˚C overnight.
sites flank the gene, allowing convenient excision. These restriction sites are
4- Isolate a single colony and grow the bacteria in TB supplemented with
compatible with many restriction sites contained in multiple cloning sites, thus
ampicillin using the FastMedia™ Amp liquid provided (see below).
5- Extract the pORF plasmid DNA using the method of your choice. Gene expression in mammalian cells. Cells may be transiently transfected with p O R F. The secreted protein may be harvested in the cell culture supernatant as Selection of bacteria with E. coli FastMedia™ Amp:
all secreted proteins in pORF possess a signal sequence. E. coli FastMedia™ Amp is a n e w, fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. mIL-15 gene may be cut out by using BspH I and Nhe I enzymes E. coli FastMedia™ Amp is a TB (liquid) or LB (solid) based medium
BspH I is compatible with Nco I and BspLU11 I.
with ampicillin, and contains stabilizers.
Nhe I is compatible with Xba I, Spe I, and Avr II. E. coli FastMedia™ Amp can be ordered separately (catalog code # fas-am-l,fas-am-s, fas-am-x). PLASMID FEATURES • EF-1α / HTLV hybrid pro m o t e r i s a composite promoter comprised of the
Elongation Factor- 1α ( E F - 1α) promoter1 and 5’ untranslated region of the
1- Pour the contents of a pouch into a clean borosilicate glass bottle or flask.
Human T-Cell Leukemia Virus (HTLV). EF-1α utilizes a type 2 promoter that
2- Add 200 ml of distilled water to the flask
encodes for a "house keeping" gene. The promoter is stronger than CMV and is
3- Heat in a microwave on MEDIUM power setting (about 400Watts), until
expressed at high levels in all cell cycles and lower levels during G0 phase. T h e
bubbles start appearing (approximately 3 minutes). Do not heat a closed
promoter is also non-tissue specific; it is highly expressed in all cell types. T h e
c o n t a i n e r. Do not autoclave FastMedia™.
R segment and part of the U5 sequence (R-U5’) of the HTLV Type 1 Long
4- Swirl gently to mix the preparation. Be careful, the bottle and media are
Terminal Repeat2 has been coupled to the EF-1α promoter to enhance stability
hot, use heatproof pads or gloves and care when handling.
of DNA and RNA. This modification not only increases steady state transcription,
5- Reheat the media for 30 seconds and gently swirl again. Repeat as
but also significantly increases translation efficiency possibly through mRNA
necessary to completely dissolve the powder into solution. But be
careful to avoid overboiling and volume loss. • Ori colE1 is a minimal E. coli origin of replication with the same activity as
6- Let agar medium cool to 45˚C before pouring plates. Let liquid media cool
• Amp (ampicillin resistance gene): The ampicillin resistance gene allows the N o t e : Do not reheat solidified FastMedia™ as the antibiotic will be
selection of bacteria carrying the pORF plasmid. permanently destroyed by the pro c e d u re .
TECHNICAL SUPPORTToll free (US): 888-457-5873Outside US: (+1) 858-457-5873E-mail: [email protected]NotI (3361) XhoI (3356) BspHI (569) pORF9-mIL-15 NheI (1093) SwaI (1355)
1 GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA
101 GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCC
201 GTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGG GGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC
301 GCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACC
401 GGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTT
BspHI (569)
501 TCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGTAGGAGGGCCATCATGAAAATTTTGAAACCATATATGAGGAAT
601 ACATCCATCTCGTGCTACTTGTGTTTCCTTCTAAACAGTCACTTTTTAACTGAGGCTGGCATTCATGTCTTCATTTTGGGCTGTGTCAGTGTAGGTCTCC
11 T h r S e r I l e S e r C y s T y r L e u C y s P h e L e u L e u A s n S e r H i s P h e L e u T h r G l u A l a G l y I l e H i s V a l P h e I l e L e u G l y C y s V a l S e r V a l G l y L e u P
701 CTAAAACAGAGGCCAACTGGATAGATGTAAGATATGACCTGGAGAAAATTGAAAGCCTTATTCAATCTATTCATATTGACACCACTTTATACACTGACAG
44 r o L y s T h r G l u A l a A s n T r p I leAspValArgTyrAspLeuGluLysI l e G l u S e r L e u I l e G l n S e r I l e H i s I leAspThrThrLeuTyrThrAspSe
801 TGACTTTCATCCCAGTTGCAAAGTTACTGCAATGAACTGCTTTCTCCTGGAATTGCAGGTTATTTTACATGAGTACAGTAACATGACTCTTAATGAAACA
77 rAspPheHisProSerCysLysValThrAlaMetAsnCysPheLeuLeuGluLeuGlnVal I leLeuHisGluTyrSerAsnMetThrLeuAsnGluThr
901 GTAAGAAACGTGCTCTACCTTGCAAACAGCACTCTGTCTTCTAACAAGAATGTAGCAGAATCTGGCTGCAAGGAATGTGAGGAGCTGGAGGAGAAAACCT111 V a l A r g A s n V a l L e u T y r L e u A l a A s n S e r T h r L e u S e r S e r A s n L y s A s n V a l A l a G l u S e r G l y C y s L y s G l u C y s G l u G l u L e u G l u G l u L y s T h r P
NheI (1093)
1001 TCACAGAGTTTTTGCAAAGCTTTATACGCATTGTCCAAATGTTCATCAACACGTCCTGACTGCACGCGAGCCTCTTCCGTGTTTCTGTTATTGCTAGCTC
144 h e T h r G l u P h e L e u G l n S e r P h e I l e A r g I l e V a l G l n M e t P h e I leAsnThrSer•••
1101 GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTG
1201 TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG
SwaI (1355)
1301 GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAGATCCATTTAAATGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGG
1401 CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA
1501 CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT
1601 CTCCCTTCGGGAAGCGTGGCGCTTTCTCATA GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC
1701 CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG
1801 GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG
1901 CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA
2001 CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCA
2101 TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATT
1 M e t S e r I l e G l n H i s P h e A r g V a l A l a L e u I l e P r o P h e P h e A l a A l a P h
2201 TTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC
17 e C y s L e u P r o V a l P h e A l a H i s P r o G l u T h r L e u V a l L y s V a l L y s A s p A l a G l u A s p G l n L e u G l y A l a A r g V a l G l y T y r I leGluLeuAspLeuAsn
2301 AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACG
51 S e r G l y L y s I l e L e u G l u S e r P h e A r g P r o G l u G l u A r g P h e P r o M e t M e t S e r T h r P h e L y s V a l L e u L e u C y s G l y A l a V a l L e u S e r A r g I leAspA
2401 CCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT
84 l a G l y G l n G l u G l n L e u G l y A r g A r g I l e H i s T y r S e r G l n A s n A s p L e u V a l G l u T y r S e r P r o V a l T h r G l u L y s H i s L e u T h r A s p G l y M e t T h r V a
2501 AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG
117 l A r g G l u L e u C y s S e r A l a A l a I leThrMetSerAspAsnThrAlaAlaAsnLeuLeuLeuThrThrI l e G l y G l y P r o L y s G l u L e u T h r A l a P h e L e u
2601 CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA
151 H i s A s n M e t G l y A s p H i s V a l T h r A r g L e u A s p A r g T r p G l u P r o G l u L e u A s n G l u A l a I leProAsnAspGluArgAspThrThrMetProValAlaM
2701 TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
184 e t A l a T h r T h r L e u A r g L y s L e u L e u T h r G l y G l u L e u L e u T h r L e u A l a S e r A r g G l n G l n L e u I l e A s p T r p M e t G l u A l a A s p L y s V a l A l a G l y P r
2801 ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGAT
217 o L e u L e u A r g S e r A l a L e u P r o A l a G l y T r p P h e I l e A l a A s p L y s S e r G l y A l a G l y G l u A r g G l y S e r A r g G l y I l e I l e A l a A l a L e u G l y P r o A s p
2901 GGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTA
251 G l y L y s P r o S e r A r g I l e V a l V a l I l e T y r T h r T h r G l y S e r G l n A l a T h r M e t A s p G l u A r g A s n A r g G l n I l e A l a G l u I l e G l y A l a S e r L e u I l e L
3001 AGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA
3101 TAATCTCATGCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCA3201 GCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTT
NotI (3361) XhoI (3356)
3301 AACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGTGACCGGATCTCGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTG3401 TGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCA3501 GGTGCCAGAACATTTCTCTATCGAA
KAVARNA - VARA 2013 LISTA HOTELURI: (CTRL + Click in lista de mai jos, pentru a accesa direct hotelul dorit) LIGHTHOUSE GOLF & SPA RESORT 5* . 2 APARTHOTEL ROYAL BAY 4* . 3 APARTHOTEL ROYAL COVE RESIDENCE 3*+ . 3 APARTHOTEL VILLA ALLEGRA 3* . 3 KALIAKRIA APARTHOTEL 3* . 4 LIGHTHOUSE GOLF & SPA RESORT 5* Localizare: Complexul este situat la 5 km nord de Balchik. In
El artículo analiza el derecho a la objeción de conciencia de los profesionales sanitarios en las sociedades multiculturales. Se sostiene la tesis de que el relativismo ético que caracteriza a estas sociedades convive con un aparentemente paradójico recorte del ejercicio del derecho a la objeción de conciencia. «Aparentemente» por-que, en el fondo, el relativismo ético tiende a la adopci�