| 质粒名称: | Nme2Cas9_pMCSG7 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 119925 |
| 存储实验室: | Erik Sontheimer |
|
载体骨架基本信息 |
|
| 载体名称: | pMCSG7 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5200 bp |
| 完整质粒大小: | 8500 bp |
| 载体类型: | 细菌表达 |
| 高拷贝/低拷贝: | 低拷贝 |
| 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 | T7 |
| 标签/融合蛋白: |
SV40 NLS（插入件上的N端子）
NLS（插入件上的C端子）
Nucleoplasmi NLS（插入C端）
|
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | TAATACGACTCACTATAGG |
| 3' 测序引物 | AGAGGCCCCAAGGGGTTATGCTAG |
|
文献引用方法 |
|
| 材料和方法部分: | Nme2Cas9_pMCSG7 was a gift from Erik Sontheimer (Addgene plasmid # 119925 ; http://n2t.net/addgene:119925 ; RRID:Addgene_119925) |
| 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 |
买家导航
Nme2Cas9pMCSG7载体基本信息
Nme2Cas9pMCSG7载体图谱质粒图谱和多克隆位点信息
Nme2Cas9pMCSG7载体简介
Nme2Cas9pMCSG7载体序列
LOCUS Nme2Cas9_pMCSG7 8691 bp ds-DNA circular SYN 22-JAN-2019
DEFINITION Nme2Cas9 bacterial expression plasmid.
ACCESSION .
VERSION .
KEYWORDS Nme2Cas9_pMCSG7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8691)
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 8691)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 22, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8691
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 963..982
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1002..1079)
/gene="lacI"
/label=lacI promoter
primer_bind 1285..1304
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 1392..1411
/label=T7
/note="T7 promoter, forward primer"
promoter 1392..1410
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1411..1435
/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)."
RBS 1450..1472
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 1483..1500
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1525..1545
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1546..1566
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4864..4890
/codon_start=1
/product="nuclear localization signal (Makkerh et al.,
1996)"
/label=NLS
/translation="PAAKKKKLD"
CDS 4903..4950
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5019..5036
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5089..5107)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5103..5150
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind complement(5274..5293)
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 5460..5563
/gene="bla"
/label=AmpR promoter
CDS 5564..6424
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5782..5801)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6595..7183
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7084..7103
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7337..7354
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 7369..7508
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(7434..7453)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7594..7616
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(7610..7801)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(8610..1001)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
ORIGIN
1 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
61 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
121 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
181 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
241 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
301 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
361 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
421 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
481 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
541 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
601 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
661 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
721 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
781 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
841 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
901 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
961 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
1021 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
1081 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
1141 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
1201 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
1261 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
1321 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
1381 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
1441 agaaataatt ttgtttaact ttaagaagga gatatacata tgcaccatca tcatcatcat
1501 tcttctggtg tagatctggg taccgagaac ctgtacttcc aatcccctaa gaagaagaga
1561 aaggtgaatg ccatggccgc cttcaagcct aacccaatca attacatcct gggactggac
1621 atcggaatcg catccgtggg atgggctatg gtggagatcg acgaggagga gaatcctatc
1681 cggctgatcg atctgggcgt gagagtgttt gagagggccg aggtgccaaa gaccggcgat
1741 tctctggcta tggcccggag actggcacgg agcgtgaggc gcctgacacg gagaagggca
1801 cacaggctgc tgagggcacg ccggctgctg aagagagagg gcgtgctgca ggcagcagac
1861 ttcgatgaga atggcctgat caagagcctg ccaaacaccc cctggcagct gagagcagcc
1921 gccctggaca ggaagctgac accactggag tggtctgccg tgctgctgca cctgatcaag
1981 caccgcggct acctgagcca gcggaagaac gagggagaga cagcagacaa ggagctgggc
2041 gccctgctga agggagtggc caacaatgcc cacgccctgc agaccggcga tttcaggaca
2101 cctgccgagc tggccctgaa taagtttgag aaggagtccg gccacatcag aaaccagagg
2161 ggcgactata gccacacctt ctcccgcaag gatctgcagg ccgagctgat cctgctgttc
2221 gagaagcaga aggagtttgg caatccacac gtgagcggag gcctgaagga gggaatcgag
2281 accctgctga tgacacagag gcctgccctg tccggcgacg cagtgcagaa gatgctggga
2341 cactgcacct tcgagcctgc agagccaaag gccgccaaga acacctacac agccgagcgg
2401 tttatctggc tgacaaagct gaacaatctg agaatcctgg agcagggatc cgagaggcca
2461 ctgaccgaca cagagagggc caccctgatg gatgagcctt accggaagtc taagctgaca
2521 tatgcccagg ccagaaagct gctgggcctg gaggacaccg ccttctttaa gggcctgaga
2581 tacggcaagg ataatgccga ggcctccaca ctgatggaga tgaaggccta tcacgccatc
2641 tctcgcgccc tggagaagga gggcctgaag gacaagaagt cccccctgaa cctgagctcc
2701 gagctgcagg atgagatcgg caccgccttc tctctgttta agaccgacga ggatatcaca
2761 ggccgcctga aggacagggt gcagcctgag atcctggagg ccctgctgaa gcacatctct
2821 ttcgataagt ttgtgcagat cagcctgaag gccctgagaa ggatcgtgcc actgatggag
2881 cagggcaagc ggtacgacga ggcctgcgcc gagatctacg gcgatcacta tggcaagaag
2941 aacacagagg agaagatcta tctgccccct atccctgccg acgagatcag aaatcctgtg
3001 gtgctgaggg ccctgtccca ggcaagaaaa gtgatcaacg gagtggtgcg ccggtacgga
3061 tctccagccc ggatccacat cgagaccgcc agagaagtgg gcaagagctt caaggaccgg
3121 aaggagatcg agaagagaca ggaggagaat cgcaaggatc gggagaaggc cgccgccaag
3181 tttagggagt acttccctaa ctttgtgggc gagccaaagt ctaaggacat cctgaagctg
3241 cgcctgtacg agcagcagca cggcaagtgt ctgtatagcg gcaaggagat caatctggtg
3301 cggctgaacg agaagggcta tgtggagatc gatcacgccc tgcctttctc cagaacctgg
3361 gacgattctt ttaacaataa ggtgctggtg ctgggcagcg agaaccagaa taagggcaat
3421 cagacaccat acgagtattt caatggcaag gacaactcca gggagtggca ggagttcaag
3481 gcccgcgtgg agacctctag atttcccagg agcaagaagc agcggatcct gctgcagaag
3541 ttcgacgagg atggctttaa ggagtgcaac ctgaatgaca ccagatacgt gaaccggttc
3601 ctgtgccagt ttgtggccga tcacatcctg ctgaccggca agggcaagag aagggtgttc
3661 gcctctaatg gccagatcac aaacctgctg aggggatttt ggggactgag gaaggtgcgg
3721 gcagagaatg acagacacca cgcactggat gcagtggtgg tggcatgcag caccgtggca
3781 atgcagcaga agatcacaag attcgtgagg tataaggaga tgaacgcctt tgacggcaag
3841 accatcgata aggagacagg caaggtgctg caccagaaga cccacttccc ccagccttgg
3901 gagttctttg cccaggaagt gatgatccgg gtgttcggca agccagacgg caagcctgag
3961 tttgaggagg ccgatacccc agagaagctg aggacactgc tggcagagaa gctgtctagc
4021 aggccagagg cagtgcacga gtacgtgacc ccactgttcg tgtccagggc acccaatcgg
4081 aagatgtctg gcgcccacaa ggacacactg agaagcgcca agaggtttgt gaagcacaac
4141 gagaagatct ccgtgaagag agtgtggctg accgagatca agctggccga tctggagaac
4201 atggtgaatt acaagaacgg cagggagatc gagctgtatg aggccctgaa ggcaaggctg
4261 gaggcctacg gaggaaatgc caagcaggcc ttcgacccaa aggataaccc cttttataag
4321 aagggaggac agctggtgaa ggccgtgcgg gtggagaaga cccaggagag cggcgtgctg
4381 ctgaataaga agaacgccta cacaatcgcc gacaatggcg atatggtgag agtggacgtg
4441 ttctgtaagg tggataagaa gggcaagaat cagtacttta tcgtgcctat ctatgcctgg
4501 caggtggccg agaacatcct gccagacatc gattgcaagg gctacagaat cgacgatagc
4561 tatacattct gtttttccct gcacaagtat gacctgatcg ccttccagaa ggatgagaag
4621 tccaaggtgg agtttgccta ctatatcaat tgcgactcct ctaacggcag gttctacctg
4681 gcctggcacg ataagggcag caaggagcag cagtttcgca tctccaccca gaatctggtg
4741 ctgatccaga agtatcaggt gaacgagctg ggcaaggaga tcaggccatg tcggctgaag
4801 aagcgcccac ccgtgcgggg cgggggtggt tcaggtggag gagggtcagg cggagggggt
4861 tcgccagcag cgaagaaaaa gaagctggat ggcgggggat ctaagcgccc agccgctaca
4921 aaaaaagcgg gacaggcgaa gaagaagaag taaaacattg gaagtggata acggatccga
4981 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
5041 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
5101 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
5161 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
5221 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
5281 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
5341 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
5401 ttagggtgat ggttcacatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
5461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
5521 aataaccctg ataaatgctt caataatttg aaaaaggaag agtatgagta ttcaacattt
5581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
5641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
5701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
5761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
5821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
5881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
5941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
6001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
6061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
6121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
6181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
6241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
6301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
6361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
6421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
6481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
6541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
6601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
6661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
6721 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
6781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
6841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
6901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
6961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
7021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
7081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
7141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
7201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ttgcgttatc
7261 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
7321 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
7381 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
7441 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
7501 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
7561 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
7621 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
7681 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
7741 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
7801 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
7861 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
7921 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
7981 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
8041 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
8101 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
8161 tgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
8221 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
8281 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
8341 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
8401 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
8461 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
8521 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
8581 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
8641 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg c
//
