| 质粒名称: | Cbh_v5 AAV-CBE N-terminal |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 137175 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | px601 |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | AAV |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | v5 AAV-CBE N-terminal; U6-protospacer，JL626 |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | Cas9 D10A |
| GenBank编号 | -- |
| 启动子 | Cbh |
| 标签/融合蛋白: | -- |
| 克隆方法: | Gibson克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | Cbh_v5 AAV-CBE N-terminal was a gift from David Liu (Addgene plasmid # 137175 ; http://n2t.net/addgene:137175 ; RRID:Addgene_137175) |
| 参考文献部分: | Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan LW, Comander J, Liu Q, Liu DR. Nat Biomed Eng. 2020 Jan;4(1):97-110. doi: 10.1038/s41551-019-0501-5. Epub 2020 Jan 14. 10.1038/s41551-019-0501-5 PubMed 31937940 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS Cbh_v5 AAV-CBE N-terminal 7691 bp ds-DNA circular SYN 28-JAN-2020
DEFINITION AAV genome: expresses the N-terminal of v5 AAV-CBE from the Cbh
promoter, and U6-sgRNA.
ACCESSION .
VERSION .
KEYWORDS Cbh_v5 AAV-CBE N-terminal
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7691)
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 7691)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 28, 2020 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7691
/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 3936..4619
/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="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
WATGLK"
CDS 4716..6431
/codon_start=1
/product="N-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(N)
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
CDS 6780..6800
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind complement(6865..6885)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 7061..7285
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
misc_RNA complement(7299..7374)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(7404..7644)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(7454..7473)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(7624..7644)
/label=hU6-F
/note="Human U6 promoter, forward primer"
repeat_region 7668..106
/label=AAV2 ITR
ORIGIN
1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc
3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
3481 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
3541 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
3601 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
3661 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
3721 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
3781 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
3841 cctgaaatca ctttttttca ggttggaccg gtgccaccat gaaacggaca gccgacggaa
3901 gcgagttcga gtcaccaaag aagaagcgga aagtctcctc agagactggg cctgtcgccg
3961 tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc tttgaccccc
4021 gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc aggcactcca
4081 tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc atcgagaagt
4141 ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg tttctgagct
4201 ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga tatccacacg
4261 tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg aataggcagg
4321 gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag caggagtccg
4381 gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac tggcctaggt
4441 acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc ctgggcctgc
4501 ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt acaatcgccc
4561 tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca ggcctgaagt
4621 ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc gagtcagcaa
4681 caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc atcggcctgg
4741 ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
4801 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
4861 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
4921 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
4981 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
5041 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
5101 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
5161 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
5221 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
5281 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
5341 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
5401 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
5461 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
5521 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
5581 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
5641 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
5701 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
5761 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
5821 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
5881 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
5941 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
6001 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
6061 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
6121 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
6181 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
6241 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
6301 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
6361 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
6421 agaaaatcga gtgcctgtcc tacgagacag agatcctgac agtggagtat ggcctgctgc
6481 caatcggcaa gatcgtggag aagaggatcg agtgtaccgt gtactctgtg gataacaatg
6541 gcaacatcta tacacagccc gtggcacagt ggcacgatag gggagagcag gaggtgttcg
6601 agtattgcct ggaggacggc agcctgatca gggcaaccaa ggaccacaag ttcatgacag
6661 tggatggcca gatgctgccc atcgacgaga ttttcgagcg ggagctggac ctgatgagag
6721 tggataacct gcctaatagc ggaggcagta aaagaacagc agacgggagt gagtttgagc
6781 ccaagaaaaa gagaaaggtg taagatctga taatcaacct ctggattaca aaatttgtga
6841 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
6901 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
6961 atcctggtta gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
7021 aggggctcgg ctgttgggca ctgacaattc cgtggtgcga ctgtgccttc tagttgccag
7081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
7141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
7201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
7261 gctggggatg cggtgggctc tatggctcga gaaaaaaagc accgactcgg tgccactttt
7321 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaaccgagac
7381 gcattaatcg tctcgggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa
7441 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc
7501 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt
7561 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga
7621 aggaatcatg ggaaataggc cctcttcctg cccgaccttg cggccgcagg aacccctagt
7681 gatggagttg g
//
