| 质粒名称: | MSP1830,pMSP1830-SaCas9KKH |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 70708 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pCAG-CFP |
| 载体出品公司: | Cepko lab (Addgene plasmid# 11179) |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 载体类型: | 哺乳细胞表达,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: | CRISPRmammalian codon-optimized KKH variant Staphylococcus aureus Cas9(E782K/N968K/R1015H)-NLS-3xFlag |
| 物种来源: | S. aureus |
| 基因突变: | E782K, N968K and R1015H in SaCas9 |
| 启动子 | CAG |
| 标签/融合蛋白: |
NLS（插接件上的C端子）
3x FLAG（插接件上的C端子）
|
| 5' 测序引物 | oBK280 (5'-CCTACAGCTCCTGGGCAACG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | MSP1830 was a gift from Keith Joung (Addgene plasmid # 70708 ; http://n2t.net/addgene:70708 ; RRID:Addgene_70708) |
| 参考文献部分: | Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, Joung JK. Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404. 10.1038/nbt.3404 PubMed 26524662 |
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 MSP1830 8097 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Human expression plasmid for SaCas9 KKH variant:
CAG-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG.
ACCESSION .
VERSION .
KEYWORDS MSP1830
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8097)
AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z,
Joung JK
TITLE Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9
by modifying PAM recognition.
JOURNAL Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404.
PUBMED 26524662
REFERENCE 2 (bases 1 to 8097)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8097
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 386..661
/label=chicken beta-actin promoter
intron 662..1670
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1594..1616
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1678..1697
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 1763..1772
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4937..4957
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4964..5029
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
primer_bind complement(5119..5138)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 5184..5239
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
primer_bind complement(5238..5257)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(5600..5616)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5600..5616)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5613..5635)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5624..5640
/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)."
promoter complement(5648..5678)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5693..5714
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5773..5968
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 5819..5954
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 5881..5900
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 5974..6108
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6011..6030)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6065..6084
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(6176..6193)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6347..6935)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6427..6446)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7106..7966)
/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 7729..7748
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7967..8071)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
1741 gcgggcccgg gatccaccgg tcgccaccat ggggaaacgg aactacatcc tggggcttga
1801 cattgggata accagcgttg gctacggaat tattgattat gagacacgcg atgtgattga
1861 cgccggggtt aggctgttca aagaggccaa cgttgaaaac aacgagggaa gacggagtaa
1921 gcgcggagca agaagactca agcgcagacg gagacatcgg attcagaggg tgaaaaagct
1981 gctcttcgat tacaatctcc tgaccgatca tagtgagctg agcggaatca acccctacga
2041 ggcgcgagtg aaagggcttt cccagaagct gtccgaagag gagttctccg ccgcgttgct
2101 gcacctggcc aaacggaggg gggttcacaa tgtaaacgaa gtggaggagg acacgggcaa
2161 tgaacttagt acgaaagaac agatcagtag gaactctaag gctctcgaag agaaatacgt
2221 cgctgagttg cagcttgaga gactgaaaaa agacggcgaa gtacgcggat ctattaatag
2281 gttcaagact tcagattacg taaaggaagc caagcagctc ctgaaagtac agaaagcgta
2341 ccatcagctc gatcagagct tcatcgatac ctacatagat ttgctggaga cacggaggac
2401 atactacgag ggcccagggg aaggatctcc ttttgggtgg aaggacatca aggaatggta
2461 cgagatgctt atgggacatt gtacatattt tccggaggag ctcaggagcg tcaagtacgc
2521 ctacaatgcc gacctgtaca atgccctcaa tgacctcaat aacctcgtga ttaccaggga
2581 cgagaacgag aagctggagt actatgaaaa gttccagatt atcgagaatg tgtttaagca
2641 gaagaagaag ccgacactta agcagattgc aaaggaaatc ctcgtgaatg aggaagatat
2701 caagggatac agagtgacaa gtacaggcaa gcccgagttc acaaatctga aggtgtacca
2761 cgatattaag gacataaccg cacgaaagga gataatcgaa aacgctgagc tcctcgatca
2821 gatcgcaaaa attcttacca tctaccagtc tagtgaggac attcaggagg aactgactaa
2881 tctgaacagt gagctcaccc aagaggaaat tgagcagatt tcaaacctga aaggctacac
2941 cgggacgcac aatctgagcc tcaaagcaat caacctcatt ctggatgaac tttggcacac
3001 aaatgacaac caaattgcca tattcaaccg cctgaaactg gtgccaaaaa aagtggatct
3061 gtcacagcaa aaggaaatcc ctacaacctt ggttgacgat tttattctgt cccccgttgt
3121 caagcggagc ttcatccagt caatcaaggt gatcaatgcc atcattaaaa aatacggatt
3181 gccaaacgat ataattatcg agcttgcacg agagaagaac tcaaaggacg cccagaagat
3241 gattaacgaa atgcagaagc gcaaccgcca gacaaacgaa cgcatagagg aaattataag
3301 aacaaccggc aaagagaatg ccaagtatct gatcgagaaa atcaagctgc acgacatgca
3361 agaaggcaag tgcctgtact ctctggaagc tatcccactc gaagatctgc tgaataatcc
3421 attcaattac gaggtggacc acatcatccc tagatccgta agctttgaca attccttcaa
3481 taacaaagtt ctggttaaac aggaggaaaa ttctaaaaaa gggaaccgga ccccgttcca
3541 gtacctgagc tccagtgaca gcaagattag ctacgagact tttaagaaac atattctgaa
3601 tctggccaaa ggcaaaggca ggatcagcaa gaccaagaag gagtacctcc tcgaagaacg
3661 cgacattaac agatttagtg tgcagaaaga tttcatcaac cgaaaccttg tcgatactcg
3721 gtacgccacg agaggcctga tgaatctcct caggagctac ttccgcgtca ataatctgga
3781 cgttaaagtc aagagcataa atgggggatt caccagcttt ctgaggagaa agtggaagtt
3841 taagaaggaa cgaaacaaag gatacaagca ccatgctgag gatgctttga tcatcgctaa
3901 cgcggacttt atctttaagg aatggaaaaa gctggataag gcaaagaaag tgatggaaaa
3961 ccagatgttc gaggagaagc aggcagagtc aatgcctgag atcgagacag agcaggaata
4021 caaggaaatt ttcatcaccc ctcatcagat taaacacata aaggacttca aagactataa
4081 atactctcat agggtggaca aaaaacccaa tcgcaagctc attaatgaca ccctgtactc
4141 aacacggaag gatgataaag gtaatacctt gattgtgaat aatcttaatg gattgtatga
4201 caaagataac gacaagctca agaagctgat caacaagtct ccagagaagc tccttatgta
4261 tcaccacgac ccacagactt atcagaaatt gaaactgatc atggagcaat acggggatga
4321 gaagaaccca ctctacaaat attatgagga aacaggtaat tacctgacca agtactccaa
4381 gaaggataac ggaccagtga tcaaaaagat aaagtactat ggcaacaaac ttaatgcgca
4441 tttggacata actgacgatt accccaattc tcgaaacaag gttgtgaagc tctccctgaa
4501 gccttataga tttgacgtgt acctggataa tggggtttat aaattcgtca ccgtgaaaaa
4561 tctggacgtg atcaaaaagg agaactatta tgaagtaaac tcaaagtgct atgaggaggc
4621 gaagaagctg aagaagatct ccaatcaggc cgagttcatc gcttccttct ataagaacga
4681 tctcatcaag atcaatggag agctttatcg cgtcattggt gtgaacaatg acttgctgaa
4741 caggatcgaa gtcaatatga tagacattac ctaccgggag tatctcgaaa acatgaatga
4801 taaacggccg cctcacatca tcaagacaat cgcatctaaa actcagtcaa taaaaaagta
4861 ctctaccgat atcctgggga atctctatga agtgaagtca aagaagcacc cacaaatcat
4921 taaaaaaggt ggatccccca agaagaagag gaaagtctcg agcgactaca aagaccatga
4981 cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagt aaagcggccg
5041 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
5101 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
5161 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
5221 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
5281 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
5341 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
5401 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
5461 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
5521 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc
5581 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
5641 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
5701 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
5761 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
5821 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
5881 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
5941 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
6001 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
6061 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
6121 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
6181 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
6241 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
6301 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
6361 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
6421 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
6481 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
6541 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
6601 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
6661 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
6721 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
6781 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
6841 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
6901 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
6961 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
7021 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
7081 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
7141 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
7201 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
7261 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
7321 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
7381 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
7441 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
7501 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
7561 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
7621 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
7681 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
7741 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
7801 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
7861 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
7921 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
7981 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
8041 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
