Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V016759 | Mm-PylRS-AF-Pyl-tRNACUA | 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
Mm-PylRS-AF is the Pyrrolysine--tRNA ligase bearing the Y306A and Y384F mutations from Methanosarcina mazei. Mm-PylRS-AF enables the encoding of various bulky non-natural lysine derivatives by UAG. This allows for the incorporation of a wider range of amino acids into proteins than what is naturally possible, thus expanding the genetic code and the potential for creating proteins with novel properties and functions. Mm-PylRS-AF ligase catalyzes the attachment of bulky non-natural lysine derivatives to tRNACUA.
- Vector Name:
- Mm-PylRS-AF-Pyl-tRNACUA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7401 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Hyg
- Promoter:
- U6
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
Mm-PylRS-AF-Pyl-tRNACUA 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
- Yanagisawa T, Kuratani M, Seki E, Hino N, Sakamoto K, Yokoyama S. Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase. Cell Chem Biol. 2019 Jul 18;26(7):936-949.e13. doi: 10.1016/j.chembiol.2019.03.008. Epub 2019 Apr 25. PMID: 31031143.
- Spence JS, He R, Hoffmann HH, Das T, Thinon E, Rice CM, Peng T, Chandran K, Hang HC. IFITM3 directly engages and shuttles incoming virus particles to lysosomes. Nat Chem Biol. 2019 Mar;15(3):259-268. doi: 10.1038/s41589-018-0213-2. Epub 2019 Jan 14. PMID: 30643282; PMCID: PMC6466627.
Mm-PylRS-AF-Pyl-tRNACUA vector Sequence
LOCUS Exported 7401 bp DNA circular SYN 17-OCT-2024
DEFINITION Exported.
ACCESSION V016759
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7401)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7401)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7401
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 47..425
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 426..629
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 860..878
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
CDS 971..994
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 995..2356
/gene="pylS"
/label=Pyrrolysine--tRNA ligase
/note="Pyrrolysine--tRNA ligase from Methanosarcina mazei
(strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 /
OCM 88). Accession#: Q8PWY1"
polyA_signal 2391..2615
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2661..3089
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3103..3432
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 3481..4503
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
polyA_signal 4636..4769
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4806..4822)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4830..4846)
/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)."
promoter complement(4854..4884)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4899..4920)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5208..5796)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5970..6827)
/label=AmpR
/note="beta-lactamase"
promoter complement(6828..6932)
/label=AmpR promoter
tRNA complement(6953..7021)
/product="tRNAPyl"
promoter complement(7030..7270)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"