Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006878 | pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase | 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
This is a 3rd generation lentiviral plasmid. It contains both Luciferase and EGFP, with puromycin selection. The promoter is CMV/TO.
- Vector Name:
- pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10490 bp
- Type:
- Mammalian Expression, Lentiviral, Luciferase
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- hPGK
pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase 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
- Lorenzatti Hiles G, Cates AL, El-Sawy L, Day KC, Broses LJ, Han AL, Briggs HL, Emamdjomeh A, Chou A, Abel EV, Liebert M, Palmbos PL, Udager AM, Keller ET, Day ML. A surgical orthotopic approach for studying the invasive progression of human bladder cancer. Nat Protoc. 2019 Mar;14(3):738-755.
pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase vector Sequence
LOCUS V006878 10490 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V006878
VERSION V006878
KEYWORDS pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 10490)
TITLE A Surgical Orthotopic Approach to Study the Invasive Progression of
Human Bladder Cancer
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 10490)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10490)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10490
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 2..304
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 305..508
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 510..528
/label="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 531..549
/gene="tetO"
/label="tet operator"
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 590..614
/label="attB1"
/note="recombination site for the Gateway(R) BP reaction"
CDS 635..1351
/label="EGFP"
/note="enhanced GFP"
CDS 1358..3007
/label="luciferase"
/note="firefly luciferase"
protein_bind complement(3024..3048)
/label="attB2"
/note="recombination site for the Gateway(R) BP reaction"
CDS 3101..3142
/codon_start=1
/product="epitope tag from simian virus 5"
/label="V5 tag"
/translation="GKPIPNPLLGLDST"
promoter 3202..3712
/label="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
CDS 3734..4330
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 4401..4989
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5069..5302
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 5374..5508
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 5535..5670
/label="SV40 ori"
/note="SV40 origin of replication"
promoter complement(5691..5709)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(5719..5736)
/label="M13 Forward"
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(5719..5735)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5728..5750)
/label="M13/pUC Forward"
/note="In lacZ gene"
rep_origin 5877..6332
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6358..6462
/label="AmpR promoter"
CDS 6463..7320
/label="AmpR"
/note="beta-lactamase"
rep_origin 7494..8082
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8236..8253
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 8370..8391
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8406..8436
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 8444..8460
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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 8449..8471
/label="M13/pUC Reverse"
/note="In lacZ gene"
primer_bind 8468..8484
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 8468..8484
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
promoter 8505..8523
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 8551..8777
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 8778..8958
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 9005..9130
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 9623..9856
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 10041..10085
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 10234..10275
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 10352..10469
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"