| 质粒名称: | pSI-Target-AID-NG |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 119861 |
| 存储实验室: | Osamu Nureki |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 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 |
| 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: | nSpCas9-NG (D10A/L1111R/D1135V/G1218R/E1219F/A1322R/R1335V/T1337R) |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 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: | pSI-Target-AID-NG was a gift from Osamu Nureki (Addgene plasmid # 119861 ; http://n2t.net/addgene:119861 ; RRID:Addgene_119861) |
| 参考文献部分: | Engineered CRISPR-Cas9 nuclease with expanded targeting space. Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, Noda T, Abudayyeh OO, Gootenberg JS, Mori H, Oura S, Holmes B, Tanaka M, Seki M, Hirano H, Aburatani H, Ishitani R, Ikawa M, Yachie N, Zhang F, Nureki O. Science. 2018 Aug 30. pii: science.aas9129. doi: 10.1126/science.aas9129. 10.1126/science.aas9129 PubMed 30166441 |
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 pSI-Target-AID-NG 8752 bp ds-DNA circular SYN 20-DEC-2018
DEFINITION Expresses Target-AID-NG
(NLS-nSpCas9-NG-NLS-SH3-3xFLAG-PmCDA1-NLS-UGI) in mammalian cells..
ACCESSION .
VERSION .
KEYWORDS pSI-Target-AID-NG
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8752)
AUTHORS Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, Noda T,
Abudayyeh OO, Gootenberg JS, Mori H, Oura S, Holmes B, Tanaka M,
Seki M, Hirano H, Aburatani H, Ishitani R, Ikawa M, Yachie N, Zhang
F, Nureki O
TITLE Engineered CRISPR-Cas9 nuclease with expanded targeting space.
JOURNAL Science. 2018 Aug 30. pii: science.aas9129. doi:
10.1126/science.aas9129.
PUBMED 30166441
REFERENCE 2 (bases 1 to 8752)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Dec 20, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8752
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(12..31)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 203..582
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 583..786
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 737..757
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 828..847
/label=T7
/note="T7 promoter, forward primer"
promoter 828..846
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 854..863
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 866..886
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3498..3509
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS 5027..5047
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5255..5320
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 5954..5974
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5981..6235
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
primer_bind complement(6258..6275)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 6264..6488
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(6559..6575)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6559..6575)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6572..6594)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 6583..6599
/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(6607..6637)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6652..6673
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6790..6807)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6961..7549)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(7041..7060)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7720..8580)
/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 8343..8362
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(8581..8685)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
61 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
121 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
181 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
241 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
301 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
361 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
421 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
481 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
541 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
601 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
661 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
721 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
781 agagctggtt tagtgaaccg tcagatccgc tagagatccg cggccgctaa tacgactcac
841 tatagggaga gccgccacca tggcaccgaa gaagaagcgt aaagtcggaa tccacggagt
901 tcctgcggca atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg
961 ttgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa
1021 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga
1081 aacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa
1141 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt
1201 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc
1261 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca
1321 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc
1381 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga
1441 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga
1501 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc
1561 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct
1621 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga
1681 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa
1741 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc
1801 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct
1861 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc
1921 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa
1981 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa
2041 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga
2101 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg
2161 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag
2221 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg
2281 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa
2341 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa
2401 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta
2461 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc
2521 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg
2581 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc
2641 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct
2701 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga
2761 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa
2821 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac
2881 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa
2941 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat
3001 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg
3061 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat
3121 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga
3181 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag
3241 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa
3301 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca
3361 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga
3421 cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt
3481 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa
3541 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt
3601 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat
3661 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc
3721 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac
3781 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga
3841 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact
3901 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga
3961 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt
4021 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat
4081 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg
4141 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa
4201 gaccgaagta cagaccggag gcttctccaa ggaaagtatc cggcccaaga ggaacagcga
4261 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgttagccc
4321 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
4381 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
4441 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
4501 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
4561 ccggttcctg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
4621 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
4681 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
4741 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
4801 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
4861 tctgggagcc cctcgggcct tcaagtactt tgacaccacc atcgaccgga aggtctaccg
4921 gtctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga
4981 aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag
5041 gaaggtgggt ggaggaggta ccggcggtgg aggctcagca gaatacgtac gagctctgtt
5101 tgacttcaat gggaatgacg aggaggatct cccctttaag aagggcgata ttctccgcat
5161 cagagataag cccgaagaac aatggtggaa tgccgaggat agcgaaggga aaaggggcat
5221 gattctggtg ccatatgtgg agaaatattc cggtgactac aaagaccatg atggggatta
5281 caaagaccac gacatcgact acaaagacga cgacgataaa tcagggatga cagacgccga
5341 gtacgtgcgc attcatgaga aactggatat ttacaccttc aagaagcagt tcttcaacaa
5401 caagaaatct gtgtcacacc gctgctacgt gctgtttgag ttgaagcgaa ggggcgaaag
5461 aagggcttgc ttttggggct atgccgtcaa caagccccaa agtggcaccg agagaggaat
5521 acacgctgag atattcagta tccgaaaggt ggaagagtat cttcgggata atcctgggca
5581 gtttacgatc aactggtatt ccagctggag tccttgcgct gattgtgccg agaaaattct
5641 ggaatggtat aatcaggaac ttcggggaaa cgggcacaca ttgaaaatct gggcctgcaa
5701 gctgtactac gagaagaatg cccggaacca gataggactc tggaatctga gggacaatgg
5761 tgtaggcctg aacgtgatgg tttccgagca ctatcagtgt tgtcggaaga ttttcatcca
5821 aagctctcat aaccagctca atgaaaaccg ctggttggag aaaacactga aacgtgcgga
5881 gaagtggaga tccgagctga gcatcatgat ccaggtcaag attctgcata ccactaagtc
5941 tccagccgtt ggtcccaaga agaaaagaaa agtcggtacc atgaccaacc tttccgacat
6001 catagagaag gaaacaggca aacagttggt catccaagag tcgatactca tgcttcctga
6061 agaagttgag gaggtcattg ggaataagcc ggaaagtgac attctcgtac acactgcgta
6121 tgatgagagc accgatgaga acgtgatgct gctcacgtca gatgccccag agtacaaacc
6181 ctgggctctg gtgattcagg actctaatgg agagaacaag atcaagatgc tataatgagt
6241 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
6301 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
6361 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
6421 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
6481 ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga cctctagcta
6541 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
6601 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag
6661 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
6721 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
6781 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
6841 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
6901 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
6961 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
7021 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
7081 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
7141 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
7201 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
7261 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
7321 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
7381 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
7441 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
7501 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
7561 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
7621 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
7681 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
7741 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
7801 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
7861 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
7921 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
7981 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
8041 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
8101 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
8161 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
8221 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
8281 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
8341 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
8401 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
8461 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
8521 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
8581 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
8641 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
8701 agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag gg
//
