TDS pBLAST42-mcs - InvivoGen

pBLAST42-mcs
A plasmid containing the IL-2 signal sequence, a multiple cloning site and the blasticidin resistance gene
Catalog # pbla42-mcs
For research use only
Version # 05H11-MT
P R O D U C T I N F O R M AT I O N
C o n t e n t :
- 20 µg of lyophilized pBLAST42-mcs plasmid DNA.
- 4 pouches of E. coli F a s t - M e d i a ® Blast (2 for agar media, 2 for liquid media).
Storage and stability:
- Products are shipped at room temperature.
- Upon receipt, resuspend lyophilized DNA and store at -20°C. Avoid repeated
freeze-thaw cycles.
- Store E. coli F a s t - M e d i a ® Blas at room temperature. Fast-Media ® pouches are
stable 18 months when stored properly.
Quality contro l :
- Plasmid DNA was prepared using an affinity column and lyophilized.
- Plasmid construct has been confirmed by restriction analysis sequencing.
G E N E R A L P R O D U C T U S E
pBLAST42-mcs is a ready-made expression vector containing the Blasticidin
resistance gene, the hybrid EF1α/ H T LV promoter and a multiple cloning site.
p B L A S T 4 2 - m c s may be used for:
Cloning in a gene of intere s t. Four unique restriction sites comprise the MCS
facilitating cloning of genes. Cloned genes will be under the control of the
E F 1α/ H T LV p r o m o t e r. Genes lacking a native signal sequence will be secreted
as the MCS is located downstream of the human IL-2 signal sequence.
As an “empty” control vector. Since pBLAST42-mcs does not contain a
therapeutic gene, it can be used in conjunction with other vectors of the pBLAST
family to serve as an experimental control.
p B L A S T is selectable with blasticidin in both E. coli and mammalian cells.
PLASMID FEAT U R E S
• EF-1α / HTLV hybrid pro m o t e r i s a composite promoter comprised of the
Elongation Factor- 1α( E F - 1α) promoter 1 and 5’untranslated region of the Human
T-Cell Leukemia Virus (HTLV). EF-1α is a ‘housekeeping’ gene ubiquitously
expressed in eukaryotic cells. The EF-1α promoter exhibits a strong activity,
higher than viral promoters and, on the contrary to the CMV p r o m o t e r, yields
persistent expression of the transgene in vivo. 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 s t a b i l i z a t i o n .
• I117 is a bacterial promoter that is spliced out as an intron in mammalian cells.
Expression of the transgene in E. coli can be further increased by the addition of
IPTG when working with bacteria that constitutively express L a c I
• SV40 polyA: 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
mRNA. The efficiency of this signal was first described by Carswell et al. 3
• SpAn: A synthetic polyadenylation site and a strong pause site are placed
downstream of the pMB1 Ori to limit transcriptional interference between both
transcriptional units. The synthetic polyA site is based on the highly eff i c i e n t
p o l y A signal of the rabbit β-globin gene 4 .
• IL2 ss: The IL2 signal sequence contains 21 amino acids and share common
characteristics with signal peptides of other secretory proteins with respect to
abundance and positions of hydrophobic amino acids. The intracellular cleavage
of the IL2 signal peptide occurs after Ser20 and leads to the secretion of the
expressed protein.
Note: The gene of interest must be cloned in frame with the IL-2ss as translation
will begin at the ATG of the IL-2ss.
• Multiple cloning site.
The MCS contains the restriction sites N r u I, A s c I, B s sH II a n d N h e I.
N ru I is compatible with any blunt-end restriction enzyme.
A s c I is compatible with A f l III, M l u I and B s sH II
B s sH II is compatible with A s c I, A f l III and M l u I .
N h e I is compatible with Xba I, Spe I, and Av r I I .
• pMB1 Ori is a minimal E. coli origin of replication with the same activity as
the longer Ori.
• SV40 prom: The Simian Virus 40 promoter allows the expression of the
blasticidin resistance gene in mammalian cells.
• Bsr (blasticidin resistance gene): The b s r gene from Bacillus cereus e n c o d e s
a deaminase that confers resistance to the antibiotic Blasticidin S. The b s r gene is
driven by the SV40 promoter in tandem with the bacterial EM7 promoter.
Therefore each pBLAST plasmid can be used to select stable mammalian cells
transfectants and E. coli t r a n s f o r m a n t s .
• bGh pAn: The bovine growth hormone (bGh) polyadenylation (pAn) signal
and a transcriptional pause are placed 3' of the blasticidin gene. The bGh pAn has
been shown to be as efficient as SV40 and HSV1tk polyadenylation signals in
many different cell types 5 . The use of bGH pAn minimizes interference and
possible recombination events with the SV40 polyadenylation signal. The pause
site prevents transcriptional interference and read-through.
R e f e re n c e s
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.
4- Levitt et al. (1989). Genes Dev. 7: 1019-1025.
5- Goodwin et al. (1992). J. Biol. Chem. 23: 16330-16334.
M E T H O D S
Plasmid re s u s p e n s i o n :
Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To
obtain a plasmid solution at 1µg/µl, resuspend the DNA in 20µl of sterile H 2 O .
Store resuspended plasmid at -20°C.
Selection of bacteria with E. coli Fast-Media Blas:
E. coli F a s t - M e d i a ® Blas is a n e w, fast and convenient way to prepare liquid and
solid media for bacterial culture by using only a microwave. E. coli F a s t - M e d i a ®
Blas is a TB (liquid) or LB (solid) based medium with blasticidin, and contains
stabilizers. E. coli F a s t - M e d i a ® Blas can be ordered separately (catalog code # fasbl-l,
fas-bl-s).
M e t h o d :
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
c o n t a i n e r. 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.
N o t e : Do not reheat solidified Fast-Media ® as the antibiotic will be permanently
d e s t royed by the pro c e d u re .
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
E-mail: info@invivogen.com
Website: www.invivogen.com
3950 Sorrento Valley Blvd. Suite A
San Diego, CA 92121 - USA

NotI (2952)
XhoI (2947)
SgfI (7)
AgeI (90)
PvuII (240)
HindIII (246)
EcoRI (2666)
hEF1/HTLV prom
EcoRV (551)
SalI (630)
BspHI (2237)
Bsr
EM7
pBLAST42-mcs
(3116 bp)
I117
hIL2ss
PvuII (659)
SgrAI (669)
EcoRI (743)
NruI (747)
AscI (753)
BssHII (754)
NheI (761)
AseI (2180)
HpaI (924)
AvrII (2110)
SV40 prom
SwaI (1022)
NcoI (2017)
pMB1 Ori
100

1
101
201
301
401
501
601
701
4
801
901
1001
1101
1201
1301
1401
1501
1601
1701
1801
1901
2001
2101
SgfI (7) AgeI (90)
GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTA
GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCC
HindIII (246)
PvuII (240)
GTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC
GCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACC
GGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTT
EcoRV (551)
TCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACgtaagtgatatctactagatttatcaaaaagagtgttgacttgtgagcgctcacaa
SalI (630) PvuII (659) SgrAI (669)
ttgatacttagattcatcgagagggacacgtcgactactaaccttcttctctttcctacagCTGAGATCACCGGCGAAGGAGGGCCACCATGTACAGGAT
1 MetTyrArgMe
BssHII (754)
AscI (753)
NruI (747)
EcoRI (743)
NheI (761)
GCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGCGAGGCGCGCCGCTAGCTCGACATGATAAGATACATTGATGAGTTTGGACA
tGlnLeuLeuSerCysI leAlaLeuSerLeuAlaLeuValThrAsnSer
AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAA
HpaI (924)
CCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAA
SwaI (1022)
CCTCTACAAATGTGGTAGATCATTTAAATGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT
TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGC
TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA
ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC
AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT
AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT
TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTAAGCTGTACACTGTGGAATGTGTG
TCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGC
TCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT
NcoI (2017)
CCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTT
AvrII (2110) AseI (2180)
TTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATcagCACGTGTTGACAATTAATCATCGGCATAGTATA
BspHI (2237)
2201 TCGGCATAGTATAATACGACAAGGTGAGGAACTAAATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGAT
1 MetLysThrPheAsnI leSerGlnGlnAspLeuGluLeuValGluValAlaThrGluLysI l
2301 CACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGC
21 eThrMetLeuTyrGluAspAsnLysHisHisValGlyAlaAlaI leArgThrLysThrGlyGluI leI leSerAlaValHisI leGluAlaTyrI leGly
2401 AGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACCCCTACTCTG
55 ArgValThrValCysAlaGluAlaI leAlaI leGlySerAlaValSerAsnGlyGlnLysAspPheAspThrI leValAlaValArgHisProTyrSerA
2501 ATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAA
88 spGluValAspArgSerI leArgValValSerProCysGlyMetCysArgGluLeuI leSerAspTyrAlaProAspCysPheValLeuI leGluMetAs
EcoRI (2666)
2601 TGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAACCTGAATTCGCTAGAGGGCCCTATTCTATAGTGTCACC
121 nGlyLysLeuValLysThrThrI leGluGluLeuI leProLeuLysTyrThrArgAsn•••
2701 TAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC
2801 CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG
NotI (2952)
XhoI (2947)
2901 GGGGAGGATTGGGAAGACAATAGCAGGCATGCGCAGGGCCCAATTGCTCGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTT
3001 TTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGA
3101 ACATTTCTCTATCGAA