| 质粒名称: | PX854 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 62886 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | PX330 |
| 载体出品公司: | Clontech |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4222 bp |
| 完整质粒大小: | 7087 bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 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 (aa573-1368) |
| 物种来源: | -- |
| 插入大小: | 2865 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AgeI(未破坏) |
| 3' 克隆位点: | NotI (未破坏) |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | PX854 was a gift from Feng Zhang (Addgene plasmid # 62886 ; http://n2t.net/addgene:62886 ; RRID:Addgene_62886) |
| 参考文献部分: | A split-Cas9 architecture for inducible genome editing and transcription modulation. Zetsche B, Volz SE, Zhang F. Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149. 10.1038/nbt.3149 PubMed 25643054 |
买家导航
PX854载体质粒基本信息
PX854质粒图谱载体图谱和PX854载体序列质粒序列多克隆位点信息
PX854质粒载体简介
PX854质粒序列载体序列
LOCUS PX854 7087 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION C-term SpCas9 piece of inducible split-5 (Cas9(C)-FKBP split-5)..
ACCESSION .
VERSION .
KEYWORDS PX854
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7087)
AUTHORS Zetsche B, Volz SE, Zhang F
TITLE A split-Cas9 architecture for inducible genome editing and
transcription modulation.
JOURNAL Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149.
PUBMED 25643054
REFERENCE 2 (bases 1 to 7087)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7087
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 1..21
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 172..191
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 440..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/label=chicken beta-actin promoter
intron 1005..1233
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1245..1254
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1257..1277
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1302..1622
/codon_start=1
/product="human FK506-binding protein FKBP12"
/label=FKBP
/translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
KLE"
CDS 1668..4052
/codon_start=1
/product="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(C)
/translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
CDS 4053..4100
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
primer_bind complement(4128..4145)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 4134..4341
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4350..4490
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4350..4479
/label=AAV2 ITR
rep_origin 4565..5020
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4652..4671)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4862..4883
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5037..5056)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5156..5178
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5216..5234)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5302..5406
/gene="bla"
/label=AmpR promoter
CDS 5407..6267
/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(5625..5644)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6438..7026
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6927..6946
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggccccaa
1261 agaagaagcg gaaggtcggt atccacggag tcccagcagc cggcgtgcag gtggagacca
1321 tcagccccgg cgacggcaga accttcccca agagaggcca gacctgcgtg gtgcactaca
1381 ccggcatgct ggaggacggc aagaagttcg acagcagcag agacagaaac aagcccttca
1441 agttcatgct gggcaagcag gaggtgatca gaggctggga ggagggcgtg gcccagatga
1501 gcgtgggcca gagagccaag ctgaccatca gccccgacta cgcctacggc gccaccggcc
1561 accccggcat catccctcct cacgccaccc tggtgttcga cgtggagctg ctgaagctgg
1621 agggtggcgg tggctcgggt ggcggtggct cgggtggcgg tggctcgtgc ttcgactccg
1681 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
1741 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
1801 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
1861 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
1921 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
1981 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
2041 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
2101 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
2161 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
2221 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
2281 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
2341 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
2401 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
2461 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
2521 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
2581 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
2641 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
2701 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
2761 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
2821 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
2881 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
2941 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
3001 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
3061 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
3121 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
3181 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
3241 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
3301 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
3361 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
3421 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
3481 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
3541 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
3601 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
3661 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
3721 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
3781 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
3841 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
3901 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
3961 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
4021 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
4081 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
4141 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
4201 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
4261 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
4321 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
4381 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
4441 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
4501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
4561 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
4621 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
4681 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
4741 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
4801 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
4861 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
4921 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
4981 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
5041 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
5101 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
5161 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
5221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
5281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
5341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
5401 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
5461 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
5521 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
5581 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
5641 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
5701 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
5761 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
5821 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
5881 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
5941 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
6001 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
6061 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
6121 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
6181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
6241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
6301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
6361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
6421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
6481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
6541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
6601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
6661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
6721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
6781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
6841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
6901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
6961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
7021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
7081 cacatgt
//
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PX854质粒载体应用举例
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