Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012143 | sortase A pentamutant (eSrtA) in pET29 | 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:
- sortase A pentamutant (eSrtA) in pET29
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5678 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T7
- 3' Primer:
- T7-term
sortase A pentamutant (eSrtA) in pET29 vector 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.
sortase A pentamutant (eSrtA) in pET29 vector Sequence
LOCUS 40924_48813 5678 bp DNA circular SYN 13-MAY-2021
DEFINITION evolved sortase A (eSrtA) pentamutant with improved kinetics and
activity.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5678)
AUTHORS Chen I, Dorr BM, Liu DR
TITLE A general strategy for the evolution of bond-forming enzymes using
yeast display.
JOURNAL Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404. doi:
10.1073/pnas.1101046108. Epub 2011 Jun 22.
PUBMED 21697512
REFERENCE 2 (bases 1 to 5678)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5678)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1101046108"; journalName: "Proc Natl Acad Sci U S A";
date: "2011-07-12- 12"; volume: "108"; issue: "28"; pages:
"11399-404"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5678
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 12..467
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(562..1374)
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin 1496..2084
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2238..2255
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(2270..2409)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 2495..2517
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2514..2702)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(3477..3498)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3514..4593)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(4594..4671)
/label=lacI promoter
primer_bind 4877..4896
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 4984..5002
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5003..5027
/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)."
RBS 5042..5064
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5522..5539
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
terminator 5606..5653
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"