| 质粒名称: | MSP680 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 65772 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | JDS246 |
| 载体出品公司: | Joung lab (Addgene Plasmid # 43861) |
| 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: | 哺乳动物表达，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: | mammalian codon-optimized Streptococcus pyogenes Cas9 (D1135E/R1335Q/T1337R)-NLS-3XFlag |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | D1135E, R1335Q, and T1337R mutations in Cas9 |
| GenBank编号 | -- |
| 启动子 | CMV & T7 |
| 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）
|
| 克隆方法: | Gibson克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | BGH-rev |
|
文献引用方法 |
|
| 材料和方法部分: | MSP680 was a gift from Keith Joung (Addgene plasmid # 65772 ; http://n2t.net/addgene:65772 ; RRID:Addgene_65772) |
| 参考文献部分: | Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369 |
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 MSP680 7614 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Human expression vector for SpCas9 EQR variant:
CMV-T7-humanSpCas9(D1135E/R1335Q/T1337R)-NLS-3xFLAG (EQR variant).
ACCESSION .
VERSION .
KEYWORDS MSP680
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7614)
AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z,
Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
JOURNAL Nature. 2015 Jun 22. doi: 10.1038/nature14592.
PUBMED 26098369
REFERENCE 2 (bases 1 to 7614)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7614
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 132..335
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 286..306
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 377..396
/label=T7
/note="T7 promoter, forward primer"
promoter 377..395
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 403..412
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 409..4512
/codon_start=1
/product="Streptococcus pyogenes Cas9 endonuclease with the
D1135V/R1335Q/T1337R mutations"
/label=Cas9 VQR
/note="has an altered recognition specificity for the
protospacer adjacent motif (PAM) (Kleinstiver et al.,
2015)"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4519..4539
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4546..4611
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 4629..4646
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(4669..4686)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 4675..4899
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(4970..4986)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4970..4986)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4983..5005)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4994..5010
/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(5018..5048)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5063..5084
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5201..5218)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5372..5960)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5452..5471)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6131..6991)
/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 6754..6773
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6992..7096)
/gene="bla"
/label=AmpR promoter
primer_bind complement(7175..7194)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 7366..131
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag
421 tattctattg gtttagacat cggcactaat tccgttggat gggctgtcat aaccgatgaa
481 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa
541 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg
601 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa
661 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc
721 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat
781 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac
841 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc
901 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg
961 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt
1021 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac
1081 ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc
1141 tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg
1201 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat
1261 cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac
1321 atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg
1381 tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct
1441 gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac
1501 ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat
1561 gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact
1621 ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga
1681 aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta
1741 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg
1801 atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa
1861 ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac
1921 gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc
1981 acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag
2041 aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg
2101 aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa
2161 gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag
2221 gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc
2281 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac
2341 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg
2401 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag
2461 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc
2521 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt
2581 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg
2641 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca
2701 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata
2761 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc
2821 caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt
2881 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa
2941 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga
3001 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg
3061 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag
3121 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc
3181 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac
3241 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg
3301 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg
3361 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta
3421 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa
3481 agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat
3541 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa
3601 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga
3661 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg
3721 ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag
3781 gactgggacc cgaaaaagta cggtggcttc gagagcccta cagttgccta ttctgtccta
3841 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg
3901 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg
3961 aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt
4021 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac
4081 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag
4141 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat
4201 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat
4261 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag
4321 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc
4381 aagtattttg acacaacgat agatcgcaaa cagtacagat ctaccaagga ggtgctagac
4441 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag
4501 cttgggggtg acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat
4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga
4621 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
4681 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
4741 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
4801 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
4861 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
4921 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
4981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
5041 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
5161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
5221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
5281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
5341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
5401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
5461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
5521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
5581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
5641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
5701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
5761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
5821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
5881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
6001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
6061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
6121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
6181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
6241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
6301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
6361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
6421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
6481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
6541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
6601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
6661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
6721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
6781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
6841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
6901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
6961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
7021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
7081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
7141 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
7201 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
7261 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
7321 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
7381 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
7441 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
7501 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
7561 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
