| 质粒名称: | pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 61592 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 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: | 哺乳细胞表达,AAV, CRISPR |
| 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: | hSaCas9 |
| 物种来源: | -- |
| 插入大小: | 3345 bp |
| 基因突变: | -- |
| GenBank编号 | CCK74173.1 |
| 启动子 | -- |
| 标签/融合蛋白: |
NLS（插入件上的N端子）
NLS（插入件上的C端子）
3xHA（插入件上的C端子）
|
| 克隆方法: | -- |
| 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: | pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA was a gift from Feng Zhang (Addgene plasmid # 61592 ; http://n2t.net/addgene:61592 ; RRID:Addgene_61592) |
| 参考文献部分: | In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 1. doi: 10.1038/nature14299. 10.1038/nature14299 PubMed 25830891 |
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 pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA 7089 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION A human codon-optimized Cas9 from Staphylococcus aureus (SaCas9)..
ACCESSION .
VERSION .
KEYWORDS pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7089)
AUTHORS Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F
TITLE In vivo genome editing using Staphylococcus aureus Cas9.
JOURNAL Nature. 2015 Apr 1. doi: 10.1038/nature14299.
PUBMED 25830891
REFERENCE 2 (bases 1 to 7089)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7089
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 16..156
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 16..145
/label=AAV2 ITR
rep_origin 231..686
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(318..337)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 528..549
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(703..722)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 822..844
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(882..900)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 968..1072
/gene="bla"
/label=AmpR promoter
CDS 1073..1933
/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(1291..1310)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2104..2692
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2593..2612
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
repeat_region 2754..2883
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
enhancer 2907..3286
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3287..3490
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 3441..3461
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 3509..3518
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3521..3541
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3566..6721
/codon_start=1
/product="Cas9 endonuclease from the Staphylococcus aureus
Type II CRISPR/Cas system"
/label=SaCas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
IKKG"
CDS 6722..6769
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6776..6856
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
primer_bind complement(6884..6901)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 6890..8
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
ORIGIN
1 gctggggagg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
61 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
121 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
181 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
241 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
301 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
361 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
421 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
481 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
541 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
601 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
661 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
721 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
781 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
841 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
901 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
961 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
1021 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
1081 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
1141 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
1201 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
1261 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
1321 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
1381 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
1441 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
1501 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
1561 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
1621 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
1681 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
1741 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
1801 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
1861 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
1921 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
1981 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
2041 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
2101 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
2161 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
2221 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
2281 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
2341 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
2401 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
2461 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
2521 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
2581 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
2641 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
2701 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
2761 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
2821 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
2881 cctgcggcct ctagactcga ggcgttgaca ttgattattg actagttatt aatagtaatc
2941 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
3001 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
3061 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
3121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
3181 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
3241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
3301 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
3361 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
3421 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
3481 aagcagagct ctctggctaa ctaccggtgc caccatggcc ccaaagaaga agcggaaggt
3541 cggtatccac ggagtcccag cagccaagcg gaactacatc ctgggcctgg acatcggcat
3601 caccagcgtg ggctacggca tcatcgacta cgagacacgg gacgtgatcg atgccggcgt
3661 gcggctgttc aaagaggcca acgtggaaaa caacgagggc aggcggagca agagaggcgc
3721 cagaaggctg aagcggcgga ggcggcatag aatccagaga gtgaagaagc tgctgttcga
3781 ctacaacctg ctgaccgacc acagcgagct gagcggcatc aacccctacg aggccagagt
3841 gaagggcctg agccagaagc tgagcgagga agagttctct gccgccctgc tgcacctggc
3901 caagagaaga ggcgtgcaca acgtgaacga ggtggaagag gacaccggca acgagctgtc
3961 caccaaagag cagatcagcc ggaacagcaa ggccctggaa gagaaatacg tggccgaact
4021 gcagctggaa cggctgaaga aagacggcga agtgcggggc agcatcaaca gattcaagac
4081 cagcgactac gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct accaccagct
4141 ggaccagagc ttcatcgaca cctacatcga cctgctggaa acccggcgga cctactatga
4201 gggacctggc gagggcagcc ccttcggctg gaaggacatc aaagaatggt acgagatgct
4261 gatgggccac tgcacctact tccccgagga actgcggagc gtgaagtacg cctacaacgc
4321 cgacctgtac aacgccctga acgacctgaa caatctcgtg atcaccaggg acgagaacga
4381 gaagctggaa tattacgaga agttccagat catcgagaac gtgttcaagc agaagaagaa
4441 gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac gaagaggata ttaagggcta
4501 cagagtgacc agcaccggca agcccgagtt caccaacctg aaggtgtacc acgacatcaa
4561 ggacattacc gcccggaaag agattattga gaacgccgag ctgctggatc agattgccaa
4621 gatcctgacc atctaccaga gcagcgagga catccaggaa gaactgacca atctgaactc
4681 cgagctgacc caggaagaga tcgagcagat ctctaatctg aagggctata ccggcaccca
4741 caacctgagc ctgaaggcca tcaacctgat cctggacgag ctgtggcaca ccaacgacaa
4801 ccagatcgct atcttcaacc ggctgaagct ggtgcccaag aaggtggacc tgtcccagca
4861 gaaagagatc cccaccaccc tggtggacga cttcatcctg agccccgtcg tgaagagaag
4921 cttcatccag agcatcaaag tgatcaacgc catcatcaag aagtacggcc tgcccaacga
4981 catcattatc gagctggccc gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga
5041 gatgcagaag cggaaccggc agaccaacga gcggatcgag gaaatcatcc ggaccaccgg
5101 caaagagaac gccaagtacc tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa
5161 gtgcctgtac agcctggaag ccatccctct ggaagatctg ctgaacaacc ccttcaacta
5221 tgaggtggac cacatcatcc ccagaagcgt gtccttcgac aacagcttca acaacaaggt
5281 gctcgtgaag caggaagaaa acagcaagaa gggcaaccgg accccattcc agtacctgag
5341 cagcagcgac agcaagatca gctacgaaac cttcaagaag cacatcctga atctggccaa
5401 gggcaagggc agaatcagca agaccaagaa agagtatctg ctggaagaac gggacatcaa
5461 caggttctcc gtgcagaaag acttcatcaa ccggaacctg gtggatacca gatacgccac
5521 cagaggcctg atgaacctgc tgcggagcta cttcagagtg aacaacctgg acgtgaaagt
5581 gaagtccatc aatggcggct tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga
5641 gcggaacaag gggtacaagc accacgccga ggacgccctg atcattgcca acgccgattt
5701 catcttcaaa gagtggaaga aactggacaa ggccaaaaaa gtgatggaaa accagatgtt
5761 cgaggaaaag caggccgaga gcatgcccga gatcgaaacc gagcaggagt acaaagagat
5821 cttcatcacc ccccaccaga tcaagcacat taaggacttc aaggactaca agtacagcca
5881 ccgggtggac aagaagccta atagagagct gattaacgac accctgtact ccacccggaa
5941 ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa
6001 tgacaagctg aaaaagctga tcaacaagag ccccgaaaag ctgctgatgt accaccacga
6061 cccccagacc taccagaaac tgaagctgat tatggaacag tacggcgacg agaagaatcc
6121 cctgtacaag tactacgagg aaaccgggaa ctacctgacc aagtactcca aaaaggacaa
6181 cggccccgtg atcaagaaga ttaagtatta cggcaacaaa ctgaacgccc atctggacat
6241 caccgacgac taccccaaca gcagaaacaa ggtcgtgaag ctgtccctga agccctacag
6301 attcgacgtg tacctggaca atggcgtgta caagttcgtg accgtgaaga atctggatgt
6361 gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct
6421 gaagaagatc agcaaccagg ccgagtttat cgcctccttc tacaacaacg atctgatcaa
6481 gatcaacggc gagctgtata gagtgatcgg cgtgaacaac gacctgctga accggatcga
6541 agtgaacatg atcgacatca cctaccgcga gtacctggaa aacatgaacg acaagaggcc
6601 ccccaggatc attaagacaa tcgcctccaa gacccagagc attaagaagt acagcacaga
6661 cattctgggc aacctgtatg aagtgaaatc taagaagcac cctcagatca tcaaaaaggg
6721 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc
6781 atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt acccatacga
6841 tgttccagat tacgcttaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
6901 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
6961 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
7021 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
7081 agcaggcat
//
