| 质粒名称: | pAAV-RSV-SpCas9 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 85450 |
| 存储实验室: | Hetian Lei |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 载体抗性: | 氨苄青霉素 |
| 载体类型: | AAV |
| 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: | pRSV |
| 启动子 | pRSV |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | XbaI (未破坏) |
| 3' 克隆位点: | AgeI (未破坏) |
| 5' 测序引物 | CGGTCTAGAaatgtagtcttatgcaatac |
| 3' 测序引物 | CGGACCGGTTTTATGTATCGAGCTAGGCAC |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-RSV-SpCas9 was a gift from Hetian Lei (Addgene plasmid # 85450 ; http://n2t.net/addgene:85450 ; RRID:Addgene_85450) |
| 参考文献部分: | The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced expression of MDM2 and proliferation and survival of cells. Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H. J Biol Chem. 2016 May 31. pii: jbc.M116.729467. 10.1074/jbc.M116.729467 PubMed 27246850 |
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 pAAV-RSV-SpCas9 7393 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Express SpCas9 in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pAAV-RSV-SpCas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7393)
AUTHORS Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H
TITLE The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced
expression of MDM2 and proliferation and survival of cells.
JOURNAL J Biol Chem. 2016 May 31. pii: jbc.M116.729467.
PUBMED 27246850
REFERENCE 2 (bases 1 to 7393)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7393
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..141
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 12..141
/label=AAV2 ITR
promoter 156..381
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
CDS 397..423
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 427..447
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 457..4557
/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 4561..4581
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 4600..4648
/note="synthetic polyadenylation signal"
repeat_region 4656..4796
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4656..4785
/label=AAV2 ITR
rep_origin 4871..5326
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4958..4977)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5168..5189
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5343..5362)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5462..5484
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5522..5540)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5608..5712
/gene="bla"
/label=AmpR promoter
CDS 5713..6573
/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(5931..5950)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6744..7332
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7233..7252
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
121 actccatcac taggggttcc tgcggcctct agaaatgtag tcttatgcaa tactcttgta
181 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
241 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
301 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
361 gtgcctagct cgatacataa aaccggtgcc accatgtacc catacgatgt tccagattac
421 gcttcgccga agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg
481 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
541 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
601 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
661 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
721 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
781 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
841 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
901 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
961 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
1021 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
1081 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
1141 cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc
1201 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
1261 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
1321 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
1381 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
1441 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
1501 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
1561 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
1621 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
1681 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
1741 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
1801 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
1861 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
1921 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
1981 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
2041 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
2101 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
2161 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
2221 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
2281 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
2341 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
2401 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
2461 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
2521 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
2581 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
2641 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
2701 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
2761 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
2821 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
2881 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
2941 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
3001 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
3061 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
3121 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
3181 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
3241 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
3301 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
3361 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
3421 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
3481 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
3541 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
3601 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
3661 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
3721 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
3781 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
3841 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
3901 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
3961 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
4021 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
4081 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
4141 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
4201 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
4261 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
4321 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
4381 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
4441 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
4501 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc
4561 cccaagaaga agagaaaggt ggaggccagc taagaattca ataaaagatc tttattttca
4621 ttagatctgt gtgttggttt tttgtgtgcg gccgcaggaa cccctagtga tggagttggc
4681 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
4741 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
4801 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
4861 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
4921 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
4981 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
5041 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
5101 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
5161 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
5221 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
5281 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
5341 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
5401 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
5461 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
5521 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
5581 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
5641 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
5701 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
5761 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
5821 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
5881 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
5941 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
6001 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
6061 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
6121 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
6181 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
6241 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
6301 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
6361 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
6421 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
6481 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
6541 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
6601 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
6661 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
6721 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
6781 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
6841 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
6901 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
6961 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
7021 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
7081 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
7141 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
7201 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
7261 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
7321 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
7381 tttgctcaca tgt
//
载体图谱质粒图谱pdf版和相关资料下载
载体质粒应用举例
- 上一篇：Bcr/Abl P210-pLEF
- 下一篇：pT3-EF1a-NICD1
