puno1-mifng - InvivoGen

pUNO1-mIFNG
An expression vector containing the mouse IFNG open reading frame
Catalog # puno1-mifng
For research use only
Version # 11E11-JC-31
PRODUCT INFORMATION
Contents:
- 1 disk of lyophilized E. coli bacteria, strain GT116
transformed by pUNO1-mIFNG
- Strain genotype is: F-, mcrA , ∆(mrr-hsdRMS-mcrBC) , Ø80lacZ∆M15 ,
∆lacX74, recA1 , endA1, ∆dcm, ∆sbcC-sbcD .
- 4 pouches of E. coli Fast-Media® Blas.
Storage and stability:
Products are shipped at room temperature. Transformed bacteria should be
stored at -20°C and are stable up to 1 year. Store E. coli Fast-Media® Blas at
room temperature. Fast-Media® pouches are stable 18 months when stored
properly.
Quality control:
Plasmid construct has been confirmed by restriction analysis and ORF
sequencing. Bacteria have been lyophilized, and their viability upon
resuspension has been verified.
GENERAL PRODUCT USE
pUNO1 are ready-made expression vectors containing a gene of interest.
pUNO1 may be used for:
Obtaining a gene to subclone into another vector. Two restriction sites
flank the gene, allowing convenient excision. These restriction sites are
compatible with many restriction sites contained in multiple cloning sites,
thus facilitating subcloning.
Stable gene expression in mammalian cells. pUNO1 plasmids can be used
directly in transfection experiments both in vitro and in vivo. pUNO1
plasmids contain the blasticidin resistance gene (bsr) driven by the CMV
promoter+enhancer in tandem with the bacterial EM7 promoter. This allows
the amplification of the plasmid in E. coli AND the selection of stable clones
in mammalian cells. pUNO1 allows a high level of expression and secretion
of the gene product.
mIFNG gene may be cut out by using AgeI and NheI enzymes.
AgeI, XmaI, BspEI, NgoMIV and SgrAI are compatible. NcoI, BspHI and
BspLU11I are compatible. NheI, XbaI, SpeI, and AvrII are compatible.
PLASMID FEATURES
• EF-1α / HTLV hybrid promoter is a composite promoter comprised of
the Elongation Factor-1α (EF-1α) promoter 1 and 5’ untranslated region of the
Human T-Cell Leukemia Virus (HTLV). EF-1α utilizes a type 2 promoter
that encodes for a "house keeping" gene. It is stronger than CMV and is
expressed at high levels in all cell cycles and lower levels during G0 phase.
The promoter is also non-tissue specific; it is highly expressed in all cell
types. The R segment and part of the U5 sequence (R-U5’) of the HTLV
Type 1 Long Terminal Repeat 2 has been coupled to the EF-1α promoter to
enhance stability of DNA and RNA. This modification not only increases
steady state transcription, but also significantly increases translation
efficiency possibly through mRNA stabilization.
• mIFNG gene:
Intronless ORF from the ATG to the stop codon.
ORF Size (bp): 468
Cloning fragment size (bp): 511
• SV40 polyA: The Simian Virus 40 late polyadenylation signal enables
efficient cleavage and polyadenylation reactions resulting in high levels of
steady-state mRNA 3 .
• pMB1 Ori is a minimal E. coli origin of replication with the same activity
as the longer Ori.
• CMV promoter & enhancer: The Cytomegalovirus promoter allows the
expression of the blasticidin resistance gene in mammalian cells.
• Bsr (blasticidin resistance gene): The bsr gene from Bacillus cereus
encodes a deaminase that confers resistance to the antibiotic Blasticidin S.
The bsr gene is driven by the CMV promoter in tandem with the bacterial
EM7 promoter. Therefore each pUNO1 plasmid can be used to select stable
mammalian cells transfectants and E. coli transformants.
• Human beta-Globin polyA: The strong human beta-globin
polyadenylation (pAn) signal is placed 3' of the blasticidin. The use of betaglobin
pAn minimizes interference and possible recombination events with
the SV40 polyadenylation signal.
References
1- Kim et al (1990). Gene 2: 217-223.
2- Takebe et al (1988). Mol. Cell Biol. 1: 466-472.
3- Carswell et al (1989). Mol. Cell Biol. 10: 4248-4258.
METHODS
Growth of pUNO1-transformed bacteria:
Use sterile conditions to do the following:
1- Resuspend the lyophilized E. coli by adding 1 ml of LB medium in the
tube containing the disk. Let sit for 5 minutes. Mix gently by inverting the
tube several times.
2- Streak bacteria taken from this suspension on an blasticidin LB agar plate
prepared with the E. coli Fast-Media® Blas agar provided (see below).
3- Place the plate in an incubator at 37˚C overnight.
4- Isolate a single colony and grow the bacteria in TB supplemented with
blasticidin using the Fast-Media® Blas liquid provided (see below).
5- Extract the pUNO1 plasmid DNA using the method of your choice.
Note: For long-term storage of the pUNO-transformed bacteria, prepare a
20% glycerol stock of the bacteria grown in the overnight liquid culture and
freeze at -80°C.
Selection of bacteria with E. coli Fast-Media Blas:
E. coli Fast-Media® Blas is a new, fast and convenient way to prepare liquid
and solid media for bacterial culture by using only a microwave.
1- Pour the contents of a 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.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
E-mail: info@invivogen.com
Website: www.invivogen.com

EagI (3517)
NotI (3516)
SwaI (3506)
PacI (3497)
SgfI (6)
PvuI (7)
MfeI (82)
EcoNI (96)
Psp1406I (203)
PvuII (239)
HindIII (245)
EcoNI (287)
Bsu36I (291)
NgoMIV (441)
hEF1-HTLV prom
KasI (535)
AgeI (552)
ApaLI (3081)
pMB1 ori
mouse IFN-gamma
EcoRV (715)
PstI (757)
BspLU11I (2767)
PacI (2757)
PstI (2750)
SdaI (2749)
pUNO1-mIFNG
(3681 bp)
NsiI (893)
PvuII (979)
Bsu36I (999)
NdeI (2571)
hCMV enh
SV40 p(A)
NheI (1063)
MscI (1069)
SnaBI (2466)
SpeI (2338)
hCMV prom
EM7
BsrS2
(h) ßglobin p(A)
HpaI (1201)
MfeI (1212)
EcoRI (1297)
100
AseI (2183)
BbsI (2121)
XmnI (2117)
StuI (1975)
BstXI (1840)
SspI (1536)
SwaI (1550)
EcoO109I (1611)

1
101
201
301
401
501
601
13
701
47
801
80
901
113
1001
147
1101
1201
1301
1401
1501
1601
1701
1801
109
1901
76
2001
43
2101
9
2201
2301
2400
2500
2600
PvuI (7)
SgfI (6)
MfeI (82) EcoNI (96)
GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACG GGTGCCTA
GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCC
HindIII (245)
Bsu36I (291)
Psp1406I (203) PvuII (239)
EcoNI (287)
GTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC
GCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACC
NgoMIV (441)
GGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTT
KasI (535) AgeI (552)
TCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGTCATCATGAACGCTACACACTGCATCTTGGCTTTGCAGCTCTT
1 M N A T H C I L A L Q L F
CCTCATGGCTGTTTCTGGCTGTTACTGCCACGGCACAGTCATTGAAAGCCTAGAAAGTCTGAATAACTATTTTAACTCAAGTGGCATAGATGTGGAAGAA
L M A V S G C Y C H G T V I E S L E S L N N Y F N S S G I D V E E
EcoRV (715) PstI (757)
AAGAGTCTCTTCTTGGATATCTGGAGGAACTGGCAAAAGGATGGTGACATGAAAATCCTGCAGAGCCAGATTATCTCTTTCTACCTCAGACTCTTTGAAG
K S L F L D I W R N W Q K D G D M K I L Q S Q I I S F Y L R L F E
NsiI (893)
TCTTGAAAGACAATCAGGCCATCAGCAACAACATAAGCGTCATTGAATCACACCTGATTACTACCTTCTTCAGCAACAGCAAGGCGAAAAAGGATGCATT
V L K D N Q A I S N N I S V I E S H L I T T F F S N S K A K K D A F
PvuII (979) Bsu36I (999)
CATGAGTATTGCCAAGTTTGAGGTCAACAACCCACAGGTCCAGCGCCAAGCATTCAATGAGCTCATCCGAGTGGTCCACCAGCTGTTGCCGGAATCCAGC
M S I A K F E V N N P Q V Q R Q A F N E L I R V V H Q L L P E S S
MscI (1069)
NheI (1063)
CTCAGGAAGCGGAAAAGGAGTCGCTGCTGATTCGGGGTGGGGAAGAGATTGTCCCAATAAGAAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGT
L R K R K R S R C •
TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACA
HpaI (1201) MfeI (1212) EcoRI (1297)
AGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAA
TTCTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAG GGGCTGTTGC
CAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTT
SspI (1536) SwaI (1550)
TAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAAT
EcoO109I (1611)
CCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTT
CTAGCTTTAGTTCCTGGTGTACTTGAGGGGGATGAGTTCCTCAATGGTGGTTTTGACCAGCTTGCCATTCATCTCAATGAGCACAAAGCAGTCAGGAGCA
141 • N R T Y K L P I L E E I T T K V L K G N M E I L V F C D P A
BstXI (1840)
TAGTCAGAGATGAGCTCTCTGCACATGCCACAGGGGCTGACCACCCTGATGGATCTGTCCACCTCATCAGAGTAGGGGTGCCTGACAGCCACAATGGTGT
Y D S I L E R C M G C P S V V R I S R D V E D S Y P H R V A V I T D
StuI (1975)
CAAAGTCCTTCTGCCCGTTGCTCACAGCAGACCCAATGGCAATGGCTTCAGCACAGACAGTGACCCTGCCAATGTAGGCCTCAATGTGGACAGCAGAGAT
F D K Q G N S V A S G I A I A E A C V T V R G I Y A E I H V A S I
GATCTCCCCAGTCTTGGTCCTGATGGCCGCCCCGACATGGTGCTTGTTGTCCTCATAGAGCATGGTGATCTTCTCAGTGGCGACCTCCACCAGCTCCAGA
I E G T K T R I A A G V H H K N D E Y L M T I K E T A V E V L E L
BbsI (2121)
XmnI (2117)
AseI (2183)
TCCTGCTGAGAGATGTTGAAGGTCTTCATGATGGCCCTCCTATAGTGAGTCGTATTATACTATGCCGATATACTATGCCGATGATTAATTGTCAAAACAG
D Q Q S I N F T K M
CGTGGATGGCGTCTCCAGCTTATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGG
SpeI (2338)
CGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTACTAGTCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGT
SnaBI (2466)
CAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCAT CATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCA
NdeI (2571)
TAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAG
TGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGG

2700
2798
2898
2998
3098
3198
3298
3398
PstI (2750)
SdaI (2749) PacI (2757) BspLU11I (2767)
GGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGC CTGCAGGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA
GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCC
GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC
ApaLI (3081)
CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT
TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG
AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC
GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAG
3498
3598
EagI (3517)
PacI (3497) SwaI (3506) NotI (3516)
TTAATTAACATTTAAATCAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCC
ATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGAA