| 质粒名称: | v3em-Cterm-PE2max-∆RNaseH-dualU6 |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 198735 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | p601 modified |
| 空载体大小: | 4967 bp |
| 完整质粒大小: | 7549bp |
| 载体类型: | AAV细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | NpuC-CtermPE2max∆RNaseH |
| 插入大小: | 2850 bp |
| 启动子: | Cbh |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | AGCGAGTCAGTGAGCGAG |
| 3’测序引物 | GTTTTCCCAGTCACGAC |
|
文献引用方法 |
|
| 材料和方法部分: |
v3em-Cterm-PE2max-∆RNaseH-dualU6 was a gift from David Liu (Addgene plasmid # 198735 ; http://n2t.net/addgene:198735 ; RRID:Addgene_198735)
|
| 参考文献部分: | Efficient prime editing in mouse brain, liver and heart with dual AAVs. Davis JR, Banskota S, Levy JM, Newby GA, Wang X, Anzalone AV, Nelson AT, Chen PJ, Hennes AD, An M, Roh H, Randolph PB, Musunuru K, Liu DR. Nat Biotechnol. 2023 May 4. doi: 10.1038/s41587-023-01758-z. 10.1038/s41587-023-01758-z PubMed 37142705 |
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 v3em-Cterm-PE2max-∆RNaseH-dualU6 7777 bp ds-DNA circular SYN 19-APR-2023
DEFINITION AAV genome encoding C-terminal PE2max and U6 expression cassettes.
ACCESSION .
VERSION .
KEYWORDS v3em-Cterm-PE2max-∆RNaseH-dualU6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7777)
TITLE Efficient AAV-Mediated In Vivo Prime Editing in Multiple Organs
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7777)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 19, 2023 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7777
/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..3632
/label=chicken beta-actin promoter
intron 3633..3860
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 3914..3934
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5129..5149
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6710..6730
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind 6750..6769
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
misc_RNA complement(6888..6963)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(6986..7299)
/label=U6 promoter
/note="RNA polymerase III promoter for mouse U6 snRNA (Das
et al., 1988)"
primer_bind complement(7047..7067)
/label=mU6-F
/note="Mouse U6 promoter, forward primer"
misc_RNA complement(7385..7460)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(7490..7730)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(7540..7559)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(7710..7730)
/label=hU6-F
/note="Human U6 promoter, forward primer"
repeat_region 7754..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 ggggggcgcg
3481 cgccaggsgg gggggggsgg ggggaggggg gggggggggs gaggsggaga ggtgsggcgg
3541 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
3601 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
3661 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
3721 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
3781 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
3841 ctgaaatcac tttttttcag gttggaccgg tgccaccatg aaacggacag ccgacggaag
3901 cgagttcgag tcaccaaaga agaagcggaa agtcatcaag attgctacac ggaaatacct
3961 gggaaagcag aacgtgtacg acatcggcgt ggagcgggat cacaacttcg ccctgaagaa
4021 tggctttatc gccagcaatt gtttcaacga aatcggcaag gctaccgcca agtacttctt
4081 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
4141 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
4201 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
4261 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
4321 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
4381 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
4441 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
4501 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
4561 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
4621 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
4681 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
4741 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
4801 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
4861 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
4921 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
4981 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
5041 acggatcgac ctgtctcagc tgggaggtga ctccggcgga agctctggtg gcagcaagcg
5101 gaccgccgac ggctctgaat tcgagagccc taagaagaaa agaaaggtga gcggaggctc
5161 tagcggcgga agcaccctga acattgaaga cgagtataga ctgcatgaaa caagcaagga
5221 acccgacgtg tccctgggct ccacctggct gtccgacttt ccccaggcct gggccgagac
5281 aggaggaatg ggcctggccg tgcggcaggc acccctgatc atccctctga aggccacctc
5341 tacacccgtg agcatcaagc agtaccctat gtctcaggag gccagactgg gcatcaagcc
5401 tcacatccag aggctgctgg accagggcat cctggtgcca tgccagagcc cctggaacac
5461 accactgctg cccgtgaaga agccaggcac caatgactat agacccgtgc aggatctgag
5521 agaggtgaac aagagggtgg aggatatcca ccccaccgtg cccaaccctt acaatctgct
5581 gtccggcctg cccccttctc accagtggta tacagtgctg gacctgaagg atgccttctt
5641 ttgtctgaga ctgcacccta ccagccagcc actgttcgcc tttgagtgga gggaccctga
5701 gatgggcatc tctggccagc tgacctggac acgcctgcct cagggcttca agaatagccc
5761 aacactgttt aacgaggccc tgcaccgcga cctggcagat ttccggatcc agcacccaga
5821 tctgatcctg ctgcagtacg tggacgatct gctgctggcc gccaccagcg agctggattg
5881 ccagcaggga acacgcgccc tgctgcagac cctgggaaac ctgggatata gggcatccgc
5941 caagaaggcc cagatctgtc agaagcaggt gaagtacctg ggctatctgc tgaaggaggg
6001 ccagagatgg ctgacagagg ccaggaagga gacagtgatg ggccagccaa cacccaagac
6061 cccaagacag ctgagggagt tcctgggcaa agcaggattt tgcaggctgt tcatcccagg
6121 attcgcagag atggcagcac ctctgtaccc actgaccaag ccgggcaccc tgtttaattg
6181 gggccctgac cagcagaagg cctatcagga gatcaagcag gccctgctga cagcaccagc
6241 cctgggcctg ccagacctga ccaagccttt cgagctgttt gtggatgaga agcagggcta
6301 cgccaagggc gtgctgaccc agaagctggg accatggaga cggcccgtgg cctatctgtc
6361 caagaagctg gacccagtgg cagcaggatg gccaccatgc ctgaggatgg tggcagcaat
6421 cgccgtgctg acaaaggatg ccggcaagct gaccatggga cagccactgg tcatcctggc
6481 accacacgca gtggaggccc tggtgaagca gcctccagat cgctggctgt ctaacgcccg
6541 gatgacacac taccaggccc tgctgctgga caccgatcgc gtgcagtttg gccctgtggt
6601 ggccctgaat ccagccaccc tgctgcctct gccagaggag ggcctgcagc acaactgtct
6661 ggactctggc ggctcaaaaa gaaccgccga cggcagcgaa ttcgagtctc ccaagaagaa
6721 gaggaaagtc taagtcgacc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
6781 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
6841 ttcaggggga gatgtgggag gttttttaaa gcaagcttgc aaaaaaagca ccgactcggt
6901 gccacttttt caagttgata acggactagc cttattttaa cttgctattt ctagctctaa
6961 aaccggcttt ttcgcgcgcg cggcaaacaa ggcttttctc caagggatat ttatagtctc
7021 aaaacacaca attactttac agttagggtg agtttccttt tgtgctgttt tttaaaataa
7081 taatttagta tttgtatctc ttatagaaat ccaagcctat catgtaaaat gtagctagta
7141 ttaaaaagaa cagattatct gtcttttatc gcacattaag cctctatagt tactaggaaa
7201 tattatatgc aaattaaccg gggcagggga gtagccgagc ttctcccaca agtctgtgcg
7261 agggggccgg cgcgggccta gagatggcgg cgtcggatcg cgaattcaaa aaaattctag
7321 ttggtttaac gcgtaactag atagaaccgc ggtgtgtttc tggagctgtt cgggctggca
7381 tcttgcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
7441 gctatttcta gctctaaaac gcgcgaacag ctccagcccg cggtgtttcg tcctttccac
7501 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
7561 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
7621 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca
7681 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccg
7741 accttgcggc cgcaggaacc cctagtgatg gagttgg
//
