Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006643 | pSL1142 (pSPIN, pBBR1 backbone) | 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
Single-plasmid V. cholerae CAST system, encodes all proteins, crRNA, and donor DNA. Entry vector encodes non-targeting crRNA, with BsaI sites for spacer cloning. pBBR1 backbone.
- Vector Name:
- pSL1142 (pSPIN, pBBR1 backbone)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 14031 bp
- Type:
- Bacterial Expression
- Replication origin:
- pBBR1 oriV
- Copy Number:
- Low Copy
- Promoter:
- J23119(SpeI)
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- cgccttcttgacgagttc
- 3' Primer:
- GCTAGTTATTGCTCAGCGG
pSL1142 (pSPIN, pBBR1 backbone) 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.
References
- Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol. 2021 Apr;39(4):480-489. doi: 10.1038/s41587-020-00745-y. Epub 2020 Nov 23.
pSL1142 (pSPIN, pBBR1 backbone) vector Sequence
LOCUS 40924_40612 14031 bp DNA circular SYN 13-MAY-2021
DEFINITION Single-plasmid V. cholerae INTEGRATE system, encodes all proteins,
crRNA, and donor DNA. Entry vector encodes non-targeting crRNA, with
BsaI sites for spacer cloning. pBBR1 backbone..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14031)
AUTHORS Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH
TITLE CRISPR RNA-guided integrases for high-efficiency, multiplexed
bacterial genome engineering.
JOURNAL Nat Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi:
10.1038/s41587-020-00745-y.
PUBMED 33230293
REFERENCE 2 (bases 1 to 14031)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14031)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi:
10.1038/s41587-020-00745-y."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14031
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(122..893)
/direction=LEFT
/label=pBBR1 oriV
/note="replication origin of the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica; requires the pBBR1
Rep protein for replication"
CDS 2373..3164
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter 3639..3673
/label=J23119 promoter
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119)"
CDS 10655..10666
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
terminator 12115..12162
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS 12317..12973
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS complement(join(13493..14031,1..121))
/codon_start=1
/label=pBBR1 Rep
/note="replication protein for the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica"
/translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"