| 质粒名称: | Nme2Cas9_pCDest |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 119923 |
| 存储实验室: | Erik Sontheimer |
|
载体骨架基本信息 |
|
| 载体名称: | pCDest |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3300 bp |
| 完整质粒大小: | 7600 bp |
| 载体类型: | 哺乳细胞表达 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Nme2Cas9 |
| 物种来源: | N. meningitidis |
| 插入大小: | 3300 bp |
| 启动子 | CMV |
| 标签/融合蛋白: | 3X HA（插入件上的C端子） |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | GGCACCGGCGGGCCCAAGAAGAA |
| 3' 测序引物 | CATGGTGGCGCCAGCCTGCTTTTTTGTAC |
|
文献引用方法 |
|
| 材料和方法部分: | Nme2Cas9_pCDest was a gift from Erik Sontheimer (Addgene plasmid # 119923 ; http://n2t.net/addgene:119923 ; RRID:Addgene_119923) |
| 参考文献部分: | A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ. Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 10.1016/j.molcel.2018.12.003. 10.1016/j.molcel.2018.12.003 PubMed 30581144 |
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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 Nme2Cas9_pCDest 7607 bp ds-DNA circular SYN 18-JAN-2019
DEFINITION Nme2Cas9 CMV-driven mammalian expression plasmid.
ACCESSION .
VERSION .
KEYWORDS Nme2Cas9_pCDest
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7607)
AUTHORS Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y,
Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
TITLE A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo
Genome Editing.
JOURNAL Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi:
10.1016/j.molcel.2018.12.003.
PUBMED 30581144
REFERENCE 2 (bases 1 to 7607)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 18, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7607
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 108..1085
/label=CMV IE94 promoter
/note="enhancer/promoter region of simian cytomegalovirus
major immediate early transcription unit IE94"
promoter 1120..1138
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1120..1137
/label=SP6
/note="SP6 promoter, forward primer"
protein_bind 1193..1217
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4488..4508
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4509..4598
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 4611..4637
/codon_start=1
/product="nuclear localization signal (Makkerh et al.,
1996)"
/label=NLS
/translation="PAAKKKKLD"
protein_bind complement(4675..4699)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(4722..4739)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 4744..4878
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4768..4787)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 4822..4841
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(4991..5011)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(4991..5009)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(5030..5046)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5030..5046)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5043..5065)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5054..5070
/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(5078..5108)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5123..5144
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5261..5278)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5432..6020)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5512..5531)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6191..7051)
/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 6814..6833
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7052..7156)
/gene="bla"
/label=AmpR promoter
rep_origin complement(7182..30)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7319..7340)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 7531..7550
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
61 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc aattcaatat
121 ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat
181 ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat
241 ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc aactggggag
301 gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc
361 atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata
421 tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat
481 gtattggccc tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc
541 cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac
601 tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg
661 tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg
721 gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat
781 tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact
841 tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat tggaagtacg
901 ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc cgcgatggct
961 gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg ggcgttccat
1021 tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat gctcgtttag
1081 ggaaccgcca ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac actatagaat
1141 acaagctact tgttcttttt gcaggatccc atcgattcga attcaaggat caacaagttt
1201 gtacaaaaaa gcaggctggc gccaccatga tggccgcctt caagcctaac ccaatcaatt
1261 acatcctggg actggacatc ggaatcgcat ccgtgggatg ggctatggtg gagatcgacg
1321 aggaggagaa tcctatccgg ctgatcgatc tgggcgtgag agtgtttgag agggccgagg
1381 tgccaaagac cggcgattct ctggctatgg cccggagact ggcacggagc gtgaggcgcc
1441 tgacacggag aagggcacac aggctgctga gggcacgccg gctgctgaag agagagggcg
1501 tgctgcaggc agcagacttc gatgagaatg gcctgatcaa gagcctgcca aacaccccct
1561 ggcagctgag agcagccgcc ctggacagga agctgacacc actggagtgg tctgccgtgc
1621 tgctgcacct gatcaagcac cgcggctacc tgagccagcg gaagaacgag ggagagacag
1681 cagacaagga gctgggcgcc ctgctgaagg gagtggccaa caatgcccac gccctgcaga
1741 ccggcgattt caggacacct gccgagctgg ccctgaataa gtttgagaag gagtccggcc
1801 acatcagaaa ccagaggggc gactatagcc acaccttctc ccgcaaggat ctgcaggccg
1861 agctgatcct gctgttcgag aagcagaagg agtttggcaa tccacacgtg agcggaggcc
1921 tgaaggaggg aatcgagacc ctgctgatga cacagaggcc tgccctgtcc ggcgacgcag
1981 tgcagaagat gctgggacac tgcaccttcg agcctgcaga gccaaaggcc gccaagaaca
2041 cctacacagc cgagcggttt atctggctga caaagctgaa caatctgaga atcctggagc
2101 agggatccga gaggccactg accgacacag agagggccac cctgatggat gagccttacc
2161 ggaagtctaa gctgacatat gcccaggcca gaaagctgct gggcctggag gacaccgcct
2221 tctttaaggg cctgagatac ggcaaggata atgccgaggc ctccacactg atggagatga
2281 aggcctatca cgccatctct cgcgccctgg agaaggaggg cctgaaggac aagaagtccc
2341 ccctgaacct gagctccgag ctgcaggatg agatcggcac cgccttctct ctgtttaaga
2401 ccgacgagga tatcacaggc cgcctgaagg acagggtgca gcctgagatc ctggaggccc
2461 tgctgaagca catctctttc gataagtttg tgcagatcag cctgaaggcc ctgagaagga
2521 tcgtgccact gatggagcag ggcaagcggt acgacgaggc ctgcgccgag atctacggcg
2581 atcactatgg caagaagaac acagaggaga agatctatct gccccctatc cctgccgacg
2641 agatcagaaa tcctgtggtg ctgagggccc tgtcccaggc aagaaaagtg atcaacggag
2701 tggtgcgccg gtacggatct ccagcccgga tccacatcga gaccgccaga gaagtgggca
2761 agagcttcaa ggaccggaag gagatcgaga agagacagga ggagaatcgc aaggatcggg
2821 agaaggccgc cgccaagttt agggagtact tccctaactt tgtgggcgag ccaaagtcta
2881 aggacatcct gaagctgcgc ctgtacgagc agcagcacgg caagtgtctg tatagcggca
2941 aggagatcaa tctggtgcgg ctgaacgaga agggctatgt ggagatcgat cacgccctgc
3001 ctttctccag aacctgggac gattctttta acaataaggt gctggtgctg ggcagcgaga
3061 accagaataa gggcaatcag acaccatacg agtatttcaa tggcaaggac aactccaggg
3121 agtggcagga gttcaaggcc cgcgtggaga cctctagatt tcccaggagc aagaagcagc
3181 ggatcctgct gcagaagttc gacgaggatg gctttaagga gtgcaacctg aatgacacca
3241 gatacgtgaa ccggttcctg tgccagtttg tggccgatca catcctgctg accggcaagg
3301 gcaagagaag ggtgttcgcc tctaatggcc agatcacaaa cctgctgagg ggattttggg
3361 gactgaggaa ggtgcgggca gagaatgaca gacaccacgc actggatgca gtggtggtgg
3421 catgcagcac cgtggcaatg cagcagaaga tcacaagatt cgtgaggtat aaggagatga
3481 acgcctttga cggcaagacc atcgataagg agacaggcaa ggtgctgcac cagaagaccc
3541 acttccccca gccttgggag ttctttgccc aggaagtgat gatccgggtg ttcggcaagc
3601 cagacggcaa gcctgagttt gaggaggccg ataccccaga gaagctgagg acactgctgg
3661 cagagaagct gtctagcagg ccagaggcag tgcacgagta cgtgacccca ctgttcgtgt
3721 ccagggcacc caatcggaag atgtctggcg cccacaagga cacactgaga agcgccaaga
3781 ggtttgtgaa gcacaacgag aagatctccg tgaagagagt gtggctgacc gagatcaagc
3841 tggccgatct ggagaacatg gtgaattaca agaacggcag ggagatcgag ctgtatgagg
3901 ccctgaaggc aaggctggag gcctacggag gaaatgccaa gcaggccttc gacccaaagg
3961 ataacccctt ttataagaag ggaggacagc tggtgaaggc cgtgcgggtg gagaagaccc
4021 aggagagcgg cgtgctgctg aataagaaga acgcctacac aatcgccgac aatggcgata
4081 tggtgagagt ggacgtgttc tgtaaggtgg ataagaaggg caagaatcag tactttatcg
4141 tgcctatcta tgcctggcag gtggccgaga acatcctgcc agacatcgat tgcaagggct
4201 acagaatcga cgatagctat acattctgtt tttccctgca caagtatgac ctgatcgcct
4261 tccagaagga tgagaagtcc aaggtggagt ttgcctacta tatcaattgc gactcctcta
4321 acggcaggtt ctacctggcc tggcacgata agggcagcaa ggagcagcag tttcgcatct
4381 ccacccagaa tctggtgctg atccagaagt atcaggtgaa cgagctgggc aaggagatca
4441 ggccatgtcg gctgaagaag cgcccacccg tgcggggcac cggcgggccc aagaagaaga
4501 ggaaggtata cccatacgat gttcctgact atgcgggcta tccctatgac gtcccggact
4561 atgcaggatc gtatccttat gacgttccag attacgctgg atccgccgct ccggcagcta
4621 agaaaaagaa actggatttc gaatccggat aagcggccgc ctcgagatat ctagacccag
4681 ctttcttgta caaagtggtt gatcctctcg agcctctaga actatagtga gtcgtattac
4741 gtagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
4801 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
4861 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
4921 gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca
4981 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
5041 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa
5101 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
5161 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
5221 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
5281 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
5341 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
5401 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
5461 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
5521 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
5581 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
5641 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
5701 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
5761 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
5821 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
5881 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
5941 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
6001 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
6061 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
6121 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
6181 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
6241 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
6301 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
6361 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
6421 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
6481 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
6541 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
6601 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
6661 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
6721 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
6781 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
6841 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
6901 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
6961 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
7021 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
7081 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
7141 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
7201 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
7261 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
7321 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
7381 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
7441 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
7501 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
7561 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccg
//
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