Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V003761 | pRep.VEE.Pac-2A-SP-GCSF | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
RepVEE-Pac-2A-SigP-GCSF (replicon) was created by replacing the GFP gene in the replicon pRepVEE-GFP with a gene cassette Pac-2A-SigP-GCSF. The gene cassette encodes a polyprotein. A gene cassette Pac-2A-SigP-GCSF was designed to direct the coexpression of an enzyme puromycin N-acetyltransferase (Pac) and a recombinant human colony-stimulating factor (G-CSF). The gene cassette Pac-2A-SigP-GCSF has the following design: its single open reading frame (ORF) encodes a polyprotein wherein the N-terminal part is the Pac, the C-terminal part is the G-CSF (with a signal peptide for secretory expression, SigP) and a central region is a protease 2A of foot-and-mouth disease virus (FMDV 2A) (Fig 2). In the replicon RepVEE.Pac-2A-SP-GCSF the gene cassette is placed downstream of the subgenomic promoter.
- Vector Name:
- pRep.VEE.Pac-2A-SP-GCSF
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10845 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Kim YG, Baltabekova AZ, Shustov AV.
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pRep.VEE.Pac-2A-SP-GCSF vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Kim YG, Baltabekova AZ, Zhiyenbay EE, Aksambayeva AS, Shagyrova ZS, Khannanov R, Ramanculov EM, Shustov AV. Recombinant Vaccinia virus-coded interferon inhibitor B18R: Expression, refolding and a use in a mammalian expression system with a RNA-vector. PLoS One. 2017 Dec 7;12(12):e0189308.
pRep.VEE.Pac-2A-SP-GCSF vector Sequence
LOCUS 40924_36733 10845 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pRep.VEE.Pac-2A-SP-GCSF, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10845)
AUTHORS Kim YG, Baltabekova AZ, Shustov AV.
TITLE Poxvirus' interferon inhibitor B18R: recombinant expression,
refolding and use in mammalian expression system based on viral
vectors
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 10845)
AUTHORS Kim YG, Baltabekova AZ, Shustov AV.
TITLE Direct Submission
JOURNAL Submitted (18-MAY-2017) National Center for Biotechnology,
Korgalzhin hwy 13/5, Astana, Akmola region 010000, Kazakhstan
REFERENCE 3 (bases 1 to 10845)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 10845)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(18-MAY-2017) National Center for Biotechnology, Korgalzhin hwy
13/5, Astana, Akmola region 010000, Kazakhstan"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..10845
/mol_type="other DNA"
/organism="synthetic DNA construct"
RBS 1536..1544
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 7576..8172
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
CDS 8842..8859
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
promoter 9083..9187
/label=AmpR promoter
CDS 9188..10045
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 10219..10807
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"