Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004073 | pPKm-145 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pPKm-145
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7573 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
- Promoter:
- EF-1α
pPKm-145 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.
pPKm-145 vector Sequence
LOCUS V004073 7573 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004073
VERSION V004073
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 7573)
AUTHORS Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao
SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
TITLE Biosynthesis of Orthogonal Molecules Using Ferredoxin and
Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB
Optogenetics
JOURNAL ACS Synth Biol (2018) In press
PUBMED 29301067
REFERENCE 2 (bases 1 to 7573)
AUTHORS Kyriakakis P.
TITLE Direct Submission
JOURNAL Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive,
PFBH room 251 MC0412, La Jolla, CA 92093, United States of America
REFERENCE 3 (bases 1 to 7573)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7573)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol (2018) In press"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251
MC0412, La Jolla, CA 92093, United States of America"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7573
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..1179
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature 1281..1867
/label="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 1887..2483
/label="PuroR"
/note="puromycin N-acetyltransferase"
LTR 2689..2922
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3079..3303
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
regulatory 3346..3355
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
promoter 3399..3503
/label="AmpR promoter"
CDS 3504..4361
/label="AmpR"
/note="beta-lactamase"
rep_origin 4535..5123
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 5282..5915
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 5962..6087
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 6580..6813
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 6998..7042
/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 7191..7232
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 7329..7446
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 7573
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"