Basic Vector Information
Expresses AcrVA5 in bacteria via T7 RNAP, for purification
pKEW212-MBP-TEV-AcrVA5 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 ℃.
pKEW212-MBP-TEV-AcrVA5 vector Sequence
LOCUS Exported 6337 bp DNA circular SYN 17-OCT-2023
DEFINITION Expresses AcrVA5 in bacteria via T7 RNAP, for purification.
ACCESSION .
VERSION .
KEYWORDS pKEW212-MBP-TEV-AcrVA5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6337)
AUTHORS Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
TITLE Systematic discovery of natural CRISPR-Cas12a inhibitors.
JOURNAL Science. 2018 Oct 12;362(6411):236-239. doi:
10.1126/science.aau5138. Epub 2018 Sep 6.
PUBMED 30190307
REFERENCE 2 (bases 1 to 6337)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aau5138"; journalName: "Science"; date:
"2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
FEATURES Location/Qualifiers
source 1..6337
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 108..130
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 150..179
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 189..1289
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind 1263..1286
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1344..1364
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1438..1458
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1465..1740
/codon_start=1
/label=anti-CRISPR N-acetyltransferase AcrVA5
/translation="MKIELSGGYICYSIEEDEVTIDMVEVTTKRQGIGSQLIDMVKDVA
REVGLPIGLYAYPQDDSISQEDLIEFYFSNDFEYDPDDVDGRLMRWS"
primer_bind complement(1867..1885)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1881..1928
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(2294..2322)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(2349..2367)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2435..2539
/gene="bla"
/label=AmpR promoter
CDS 2540..3400
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(2758..2777)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3571..4159
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4060..4079
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4313..4330
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4345..4484
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4410..4429)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4570..4592
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4586..4777)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind 6235..6254
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
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