Basic Vector Information
pBK-EQR-BE3 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 ℃.
pBK-EQR-BE3 vector Sequence
LOCUS 40924_6581 8532 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses EQR-BE3 in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8532)
AUTHORS Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR
TITLE Increasing the genome-targeting scope and precision of base editing
with engineered Cas9-cytidine deaminase fusions.
JOURNAL Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803.
PUBMED 28191901
REFERENCE 2 (bases 1 to 8532)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8532)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8532
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 132..335
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 377..395
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 409..1095
/codon_start=1
/product="cytidine deaminase (C to U editing enzyme) from
rat"
/label=APOBEC-1
/note="can use ssDNA as a substrate (Komor et al., 2016)"
/translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
LWATGLK"
CDS 1144..5244
/codon_start=1
/label=Cas9 VQR
/note="Streptococcus pyogenes Cas9 endonuclease with the
D1135V/R1335Q/T1337R mutations"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5257..5505
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 5518..5538
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5547..5564
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
polyA_signal 5593..5817
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(5888..5904)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5912..5928)
/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(5936..5966)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5981..6002)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6119..6136)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6290..6878)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7052..7909)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7910..8014)
/label=AmpR promoter
primer_bind complement(8093..8112)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 8284..8532
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
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