Basic Vector Information
Cbh_v5 AAV-CBE C-terminal 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 ℃.
Cbh_v5 AAV-CBE C-terminal vector Sequence
LOCUS Exported 7658 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION AAV genome: expresses the C-terminal of v5 AAV-CBE from the Cbh
promoter, and U6-sgRNA.
ACCESSION .
VERSION .
KEYWORDS Cbh_v5 AAV-CBE C-terminal
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7658)
AUTHORS Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan
LW, Comander J, Liu Q, Liu DR
TITLE Cytosine and adenine base editing of the brain, liver, retina, heart
and skeletal muscle of mice via adeno-associated viruses.
JOURNAL Nat Biomed Eng. 2020 Jan;4(1):97-110. doi:
10.1038/s41551-019-0501-5. Epub 2020 Jan 14.
PUBMED 31937940
REFERENCE 2 (bases 1 to 7658)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/s41551-019-0501-5"; journalName: "Nat Biomed Eng"; date:
"2020-01"; volume: "4"; issue: "1"; pages: "97-110"
FEATURES Location/Qualifiers
source 1..7658
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(125..142)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(125..141)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(134..156)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 283..738
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(370..389)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 580..601
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 764..868
/gene="bla"
/label=AmpR promoter
CDS 869..1729
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(1087..1106)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1900..2488
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2389..2408
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2642..2659
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2776..2797
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2812..2842
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2850..2866
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
primer_bind 2855..2877
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 2874..2890
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2874..2890
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
repeat_region 2917..3046
/label=AAV2 ITR
enhancer 3069..3354
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 3356..3633
/label=chicken beta-actin promoter
intron 3634..3861
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 3915..3935
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4041..6425
/codon_start=1
/product="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(C)
/translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
CDS 6456..6704
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 6747..6767
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind complement(6832..6852)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 7028..7252
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
misc_RNA complement(7266..7341)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(7371..7611)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(7421..7440)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(7591..7611)
/label=hU6-F
/note="Human U6 promoter, forward primer"
repeat_region join(7635..7658,1..106)
/label=AAV2 ITR
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