Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V016432 | pT-GTW6-CMV-mKate | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pT-GTW6-CMV-mKate
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4102 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Frei T, Cella F, Tedeschi F, Gutierrez J
- Promoter:
- CMV
- Growth Strain(s):
- Top10
pT-GTW6-CMV-mKate 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.
pT-GTW6-CMV-mKate vector Sequence
LOCUS 62056_20340 4102 bp DNA circular SYN 17-NOV-2020
DEFINITION Cloning vector pT-GTW6-CMV-mKate, complete sequence.
ACCESSION MT891367
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4102)
AUTHORS Frei T, Cella F, Tedeschi F, Gutierrez J, Stan GB, Khammash M,
Siciliano V.
TITLE Characterization and mitigation of gene expression burden in
mammalian cells
JOURNAL Nat Commun 11 (1), 4641 (2020)
PUBMED 32934213
REFERENCE 2 (bases 1 to 4102)
AUTHORS Frei T, Cella F, Tedeschi F, Gutierrez J, Stan G-B., Khammash M,
Siciliano V.
TITLE Direct Submission
JOURNAL Submitted (15-AUG-2020) Synthetic and Systems Biology for
Biomedicine, Italian Institute of Technology-IIT, Largo Barsanti e
Martteucci 53, Naples, Campania 80125, Italy
REFERENCE 3 (bases 1 to 4102)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Commun"; date: "2020"; volume: "11"; issue: "1"; pages: "4641"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-AUG-2020) Synthetic and Systems Biology for Biomedicine, Italian
Institute of Technology-IIT, Largo Barsanti e Martteucci 53, Naples,
Campania 80125, Italy"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..4102
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 59..79
/label=attB4
/note="core recombination site for the Gateway(R) BP
reaction"
enhancer 151..454
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 455..658
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 689..713
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 748..765
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 766..1473
/codon_start=1
/label=PAmKate
/note="monomeric photoactivatable red fluorescent protein
(Gunewardene et al., 2011)"
/translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
IKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDM
ALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVA
RYCDLPSKLGHKLN"
protein_bind complement(1506..1530)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
polyA_signal 1659..1780
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1787..2242)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2269..2373
/label=AmpR promoter
CDS 2374..3231
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3405..3993
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"