Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V010409 | pLX303 | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pLX303
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 9335 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, Lubonja
- Copy Number:
- High copy number
- Promoter:
- hPGK
pLX303 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.
pLX303 vector Sequence
LOCUS pLX303. 9335 bp DNA circular SYN 01-JAN-1980
DEFINITION Lentiviral Gateway(R) destination vector with a blasticidin
resistance marker, for strong expression and improved plasmid yield
and viral titer.
ACCESSION .
VERSION .
KEYWORDS pLX303
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9335)
AUTHORS Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, Lubonja
R, Thomas SR, Alkan O, Bhimdi T, Green TM, Johannessen CM, Silver
SJ, Nguyen C, Murray RR, Hieronymus H, Balcha D, Fan C, Lin C,
Ghamsari L, Vidal M, Hahn WC, Hill DE, Root DE.
TITLE A public genome-scale lentiviral expression library of human ORFs.
JOURNAL Nat. Methods 2011;8:659-61.
PUBMED 21706014
REFERENCE 2 (bases 1 to 9335)
AUTHORS Root Lab
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9335)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Methods"; date: "2011"; volume: "8"; pages: "659-61"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9335
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 65..368
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 369..572
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 595..719
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 744..774
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 828..1484
/label=CmR
/note="chloramphenicol acetyltransferase"
CDS 1829..2131
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
protein_bind complement(2175..2299)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
misc_feature 2301..2309
/label=stop codons
/note="stop codons"
misc_feature 2342..2930
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 3002..3235
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3313..3447
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 3474..3609
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(3630..3648)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3658..3674)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3816..4271
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4297..4401
/label=AmpR promoter
CDS 4402..5259
/label=AmpR
/note="beta-lactamase"
rep_origin 5433..6021
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 6309..6330
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6345..6375
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6383..6399
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 6407..6423
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 6444..6462
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6490..6716
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6717..6897
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6944..7069
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7562..7795
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7980..8024
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
promoter 8199..8699
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 8711..9106
/label=BSD
/note="blasticidin S deaminase"
misc_feature 9166..9283
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"