Basic Vector Information
- Vector Name:
- BPK2014-AaCas12b
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 7180 bp
- Type:
- Bacterial Expression
- Replication origin:
- p15A ori
- Copy Number:
- Low Copy
- Cloning Method:
- Restriction Enzyme
BPK2014-AaCas12b vector Vector Map
Plasmid Resuspension 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 ℃.
BPK2014-AaCas12b vector Sequence
LOCUS 40924_355 7180 bp DNA circular SYN 13-MAY-2021
DEFINITION E. coli expression, protein purification.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7180)
AUTHORS Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
TITLE Repurposing CRISPR-Cas12b for mammalian genome engineering.
JOURNAL Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3.
eCollection 2018.
PUBMED 30510770
REFERENCE 2 (bases 1 to 7180)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7180)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell
Discov."; date: "2018-11-27"; pages: "
10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7180
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 12..36
/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 51..73
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 83..103
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 3503..3529
/codon_start=1
/label=9xHis
/note="9xHis affinity tag"
/translation="HHHHHHHHH"
promoter 3601..3619
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
terminator 3641..3688
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(3912..4568)
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(4569..4671)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
primer_bind complement(5063..5080)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5197..5742)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."
protein_bind complement(5933..5954)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(5970..7049)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(7050..7127)
/label=lacI promoter
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