| 质粒名称: | PX551 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 60957 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 载体抗性: | 氨苄西林 |
| 空载体大小: | 2905 bp |
| 完整质粒大小: | 7400bp |
| 载体类型: | 哺乳动物表达，AAV，CRISPR |
| 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: | SpCas9 |
| 备用名称: | S. pyogenes Cas9 nuclease、Cas9、 |
| 物种来源: | 合成的 |
| 插入大小: | 4200 bp |
| 启动子 | pMecp2 |
| 标签/融合蛋白: | HA（插入件上的N端子） |
| 克隆方法: | 限制性酶 |
| 5' 克隆位点: | Agei（未销毁） |
| 3' 克隆位点: | Ecori（未销毁） |
| 5' 测序引物 | AATGGGGTCCGCCTCTTTTCC |
| 3' 测序引物 | cttagctggcctccacctttctcttc |
|
文献引用方法 |
|
| 材料和方法部分: | PX551 was a gift from Feng Zhang (Addgene plasmid # 60957 ; http://n2t.net/addgene:60957 ; RRID:Addgene_60957) |
| 参考文献部分: | In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F. Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055. 10.1038/nbt.3055 PubMed 25326897 |
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 PX551 7389 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION pAAV-pMecp2-SpCas9-spA (AAV-SpCas9). AAV plasmid expressing Cas9 in
neurons under control of truncated mecp2 promoter..
ACCESSION .
VERSION .
KEYWORDS PX551
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7389)
AUTHORS Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur
M, Zhang F
TITLE In vivo interrogation of gene function in the mammalian brain using
CRISPR-Cas9.
JOURNAL Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055.
PUBMED 25326897
REFERENCE 2 (bases 1 to 7389)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7389
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 106..334
/label=Mecp2 promoter
/note="promoter for selective expression in neurons (Adachi
et al., 2005; Gray et al., 2011)"
CDS 350..376
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 380..400
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 410..4510
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 4514..4534
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 4553..4601
/note="synthetic polyadenylation signal"
repeat_region 4609..4749
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4609..4738
/label=AAV2 ITR
rep_origin 4824..5279
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4911..4930)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5121..5142
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5296..5315)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5415..5437
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5475..5493)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5561..5665
/gene="bla"
/label=AmpR promoter
CDS 5666..6526
/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(5884..5903)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6697..7285
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7186..7205
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
repeat_region 7347..87
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
1 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
61 tggccaactc catcactagg ggttcctgcg gcctctagaa agcttagctg aatggggtcc
121 gcctcttttc cctgcctaaa cagacaggaa ctcctgccaa ttgagggcgt caccgctaag
181 gctccgcccc agcctgggct ccacaaccaa tgaagggtaa tctcgacaaa gagcaagggg
241 tggggcgcgg gcgcgcaggt gcagcagcac acaggctggt cgggagggcg gggcgcgacg
301 tctgccgtgc ggggtcccgg catcggttgc gcgcaccggt gccaccatgt acccatacga
361 tgttccagat tacgcttcgc cgaagaaaaa gcgcaaggtc gaagcgtccg acaagaagta
421 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
481 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
541 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
601 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
661 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
721 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
781 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
841 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
901 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
961 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
1021 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
1081 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
1141 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
1201 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
1261 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
1321 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
1381 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
1441 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
1501 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
1561 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
1621 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
1681 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
1741 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
1801 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
1861 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
1921 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
1981 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
2041 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
2101 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
2161 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
2221 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
2281 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
2341 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
2401 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
2461 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
2521 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
2581 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
2641 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
2701 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
2761 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
2821 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
2881 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
2941 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
3001 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
3061 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
3121 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
3181 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
3241 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
3301 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
3361 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
3421 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
3481 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
3541 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
3601 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
3661 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
3721 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
3781 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
3841 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
3901 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
3961 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
4021 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
4081 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
4141 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
4201 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
4261 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
4321 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
4381 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
4441 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
4501 gggaggcgac agccccaaga agaagagaaa ggtggaggcc agctaagaat tcaataaaag
4561 atctttattt tcattagatc tgtgtgttgg ttttttgtgt gcggccgcag gaacccctag
4621 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
4681 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
4741 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
4801 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
4861 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
4921 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
4981 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
5041 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
5101 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
5161 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
5221 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
5281 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
5341 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
5401 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
5461 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
5521 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
5581 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
5641 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
5701 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
5761 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
5821 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
5881 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
5941 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
6001 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
6061 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
6121 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
6181 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
6241 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
6301 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
6361 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
6421 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
6481 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
6541 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
6601 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
6661 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
6721 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
6781 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
6841 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
6901 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
6961 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
7021 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
7081 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
7141 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
7201 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
7261 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
7321 cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga
7381 ggccgcccg
//
