| 质粒名称: | MLM3613 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 42251 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | -- |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | CRISPR；斑马鱼表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素,100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | XL1 Blue |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | Streptococcus pyogenes Cas9,Cas9-001 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | CMV |
| 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 | CMV-F |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | MLM3613 was a gift from Keith Joung (Addgene plasmid # 42251 ; http://n2t.net/addgene:42251 ; RRID:Addgene_42251) |
| 参考文献部分: | Efficient genome editing in zebrafish using a CRISPR-Cas system. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501. 10.1038/nbt.2501 PubMed 23360964 |
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 MLM3613 7554 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses Cas9 nuclease (Streptococcus pyogenes) from CMV and T7
promoters.
ACCESSION .
VERSION .
KEYWORDS MLM3613
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7554)
AUTHORS Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT,
Yeh JR, Joung JK
TITLE Efficient genome editing in zebrafish using a CRISPR-Cas system.
JOURNAL Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.
PUBMED 23360964
REFERENCE 2 (bases 1 to 7554)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7554
/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="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
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 4569..4586
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(4609..4626)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 4615..4839
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(4910..4926)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4910..4926)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4923..4945)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4934..4950
/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(4958..4988)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5003..5024
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5141..5158)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5312..5900)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5392..5411)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6071..6931)
/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 6694..6713
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6932..7036)
/gene="bla"
/label=AmpR promoter
primer_bind complement(7115..7134)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 7306..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 ggataagaaa
421 tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa
2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
4501 ctaggaggtg acggttctcc caagaagaag aggaaagtct cgagcggtgg agctgcagga
4561 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
4621 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
4681 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
4741 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
4801 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
4861 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
4921 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
4981 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
5041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
5101 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
5161 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
5221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
5281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
5341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
5401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
5461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
5521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
5581 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
5641 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
5701 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
5761 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
5821 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
5881 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
5941 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
6001 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
6061 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
6121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
6181 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
6241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
6301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
6361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
6421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
6481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
6541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
6601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
6661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
6721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
6781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
6841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
6901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
6961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
7021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
7081 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
7141 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
7201 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
7261 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
7321 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
7381 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
7441 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
7501 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
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