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Technology at Applied
Viromics
Applied Viromics uses its advanced platform
technology to produce adenovirus, AAV, retrovirus, and baculovirus.
Once the gene of interest is acquired, it can be cloned into a series
of shuttle plasmids for each viral vector. This stream-lined strategy gives
our customers advantages of having multiple choices with less effort. We
believe when you progress deep down into your project, you may want
to test your favorite genes on different systems. No matter it is
different cell line, animal model, or biochemical study with
purified proteins, we have a type of viral vector ready to go.
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The recombinant gene needs to be cloned into a shuttle
plasmid before viral vector can be generated. At Applied Viromics, we
have prepared various versions of cloning plasmids to meet your special
needs. We have constructs with different promoters, including
inducible ones, with N-terminal tag sequence to allow fusion protein
expression, and with marker gene cassette for selection.
All above shuttle plasmids are extensively tested and work great. We
are glad to subclone your gene of interest into the shuttle plasmids
that you choose. We also accept shuttle plasmids prepare yourself based on compatibility
information provided on this website. Please contact us if you want to
use shuttle plasmid of yourselves or your own expression cassette. Features of our shuttle
plasmids for individual viral vector types are listed below:
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Plasmid Map
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Features
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 Restriction sites: A (Sfi I), B (Nhe I), C (Asi SI) D
(Asc I), E (Sal I), F (Bgl II), G (Pac I)
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1. LITR: Left inverted terminal repeat
2. y:
encapsidation signal
3. P: promoter; A(n): poly-A sequence
4. lacZa mini-cassette facilitates
cloning
5. Plasmid linearized by Pac I digestion.
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Restriction sites: Same as in adenovirus shuttle plasmid
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1. Whole expression cassette removable by
Sal I - Bgl II digestion
2. Gene of interested can be cloned in
2xSfi I sites (ligated with Sfi I adaptor) or in Nhe I - Asi SI/Asc I
sites.
3. Max insert size between ITRs: 5kb
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 Restriction sites: A (Sfi I), B
(Avr II), C (Mlu I), D (Asi SI), E (Sal I), F (Bgl II)
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1. Use enzymes with compatible ends (Avr
II vs. Nhe I, and Mlu I vs Asc I)
2. Expression cassette in opposite orientation
of the LTR to accommodate introns
3. Provide neomycin selection marker
4. Same flanking restriction sites (Sal I
- Bgl II) allow one expression cassette to be cloned in all three
shuttle plasmids together
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Restriction sites: A (Sfi I), B (Nhe I), C (Asc I), D (Asi SI)
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1. Use strong Polyhedrin promoter
2. Two-region homologous recombination
3. All 4 shuttle plasmids have the same multi-cloning sites
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Insert
size limitation
Viral capsid can only pack its genome up to
certain size. For recombinant viral vectors, some viral sequences
are removed to accommodate the expression cassette for the gene of
interest. Thus the size of the gene, together with its promoter and
poly-A sequence, can not exceed certain limit. Our scientists at
Applied Viromics will further discuss with you when we work on your
gene for shuttle plasmid construction. Table below gives guideline
for this matter:
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| Vector Type
Max. Insert Size
Gene Size Limit(*)
Adeno (-E1,
-E3)
6.5
kb
4.0 - 5.3 kb
AAV
4.7
kb
2.2 - 3.5 kb
Retro (MMLV)
6.8
kb
4.3 - 5.6 kb
Baculo (AcMNPV)
no known upper size limit
(*) This limit applies to genes cloned into
Applied Viromics' shuttle plasmids. |
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