| 质粒名称: | EC2_3_dCas9_Mxi1_sgRNA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 163708 |
| 存储实验室: | Yvonne Nygård |
|
载体骨架基本信息 |
|
| 载体名称: | MoClo-YTK |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | 9502 bp |
| 载体修饰: | -- |
| 载体类型: | 酵母表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 卡那霉素50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dCas9,Nuclease-deficient Cas9 |
| 物种来源: | Streptococcus pyogenes |
| 插入大小: | 4101 bp |
| 基因突变: | Point mutations D10A and H840A |
| GenBank编号 | -- |
| 启动子: | ScTEF1 |
| 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 | Golden Gate Assembly |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | EC2_3_dCas9_Mxi1_sgRNA was a gift from Yvonne Nygård (Addgene plasmid # 163708 ; http://n2t.net/addgene:163708 ; RRID:Addgene_163708) |
| 参考文献部分: | A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12. Camara E, Lenitz I, Nygard Y. Sci Rep. 2020 Sep 3;10(1):14605. doi: 10.1038/s41598-020-71648-w. 10.1038/s41598-020-71648-w PubMed 32884066 |
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 EC2_3_dCas9_Mxi1_sgRNA 9502 bp ds-DNA circular SYN 01-NOV-2021
DEFINITION Yeast low copy plasmid with dCas9, sgRNA expression cassette and
Mxi1 transcriptional repressor.
ACCESSION .
VERSION .
KEYWORDS EC2_3_dCas9_Mxi1_sgRNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9502)
AUTHORS Camara E, Lenitz I, Nygard Y
TITLE A CRISPR activation and interference toolkit for industrial
Saccharomyces cerevisiae strain KE6-12.
JOURNAL Sci Rep. 2020 Sep 3;10(1):14605. doi: 10.1038/s41598-020-71648-w.
PUBMED 32884066
REFERENCE 2 (bases 1 to 9502)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Nov 1, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9502
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 328..735
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
CDS 758..778
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4886..4906
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4913..5035
/codon_start=1
/product="transcriptional repressor domain from the mouse
Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
/label=Mxi1
/translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
CDS 5036..5056
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
misc_RNA 5618..5693
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 6204..7013
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
terminator 7018..7215
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
misc_feature 7220..7723
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
CDS complement(7767..8582)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 8574..8593
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 8807..9395
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 9296..9315
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ccctgaattc gcatctagac tgaactggcc gataattgca gacgaacgcc ttgccaacag
61 ggagttcttc agagacatgg aggctcaaaa cgaaattatt gacagcctag acatcaatag
121 tcatacaaca gaaagcgacc acccaacttt ggctgataat agcgtataaa caatgcatac
181 tttgtacgtt caaaatacaa tgcagtagat atatttatgc atattacata taatacatat
241 cacataggaa gcaacaggcg cgttggactt ttaattttcg aggaccgcga atccttacat
301 cacacccaat cccccacaag tgatccccca cacaccatag cttcaaaatg tttctactcc
361 ttttttactc ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc
421 aagcacagca tactaaattt cccctctttc ttcctctagg gtgtcgttaa ttacccgtac
481 taaaggtttg gaaaagaaaa aagacaccgc ctcgtttctt tttcttcgtc gaaaaaggca
541 ataaaaattt ttatcacgtt tctttttctt gaaaattttt ttttttgatt tttttctctt
601 tcgatgacct cccattgata tttaagttaa taaacggtca tcaatttctc aagtttcagt
661 ttcatttttc ttgttctatt acaacttttt ttacttcttg ctcattagaa agaaagcata
721 gcaatctaat ctaagtttta attacaaaag atctatgcca aagaagaaga gaaaggtagg
781 cagcgacaag aagtattcta tcggactggc catcgggact aatagcgtcg ggtgggccgt
841 gatcactgac gagtacaagg tgccctctaa gaagttcaag gtgctcggga acaccgaccg
901 gcattccatc aagaaaaatc tgatcggagc tctcctcttt gattcagggg aaaccgctga
961 agcaacccgc ctcaagcgga ctgctagacg gcggtacacc aggaggaaga accggatttg
1021 ttaccttcaa gagatattct ccaacgaaat ggcaaaggtc gacgacagct tcttccatag
1081 gctggaagaa tcattcctcg tggaagagga taagaagcat gaacggcatc ccatcttcgg
1141 taatatcgtc gacgaggtgg cctatcacga gaaataccca accatctacc atcttcgcaa
1201 aaagctggtg gactcaaccg acaaggcaga cctccggctt atctacctgg ccctggccca
1261 catgatcaag ttcagaggcc acttcctgat cgagggcgac ctcaatcctg acaatagcga
1321 tgtggataaa ctgttcatcc agctggtgca gacttacaac cagctctttg aagagaaccc
1381 catcaatgca agcggagtcg atgccaaggc cattctgtca gcccggctgt caaagagccg
1441 cagacttgag aatcttatcg ctcagctgcc gggtgaaaag aaaaatggac tgttcgggaa
1501 cctgattgct ctttcacttg ggctgactcc caatttcaag tctaatttcg acctggcaga
1561 ggatgccaag ctgcaactgt ccaaggacac ctatgatgac gatctcgaca acctcctggc
1621 ccagatcggt gaccaatacg ccgacctttt ccttgctgct aagaatcttt ctgacgccat
1681 cctgctgtct gacattctcc gcgtgaacac tgaaatcacc aaggcccctc tttcagcttc
1741 aatgattaag cggtatgatg agcaccacca ggacctgacc ctgcttaagg cactcgtccg
1801 gcagcagctt ccggagaagt acaaggaaat cttctttgac cagtcaaaga atggatacgc
1861 cggctacatc gacggaggtg cctcccaaga ggaattttat aagtttatca aacctatcct
1921 tgagaagatg gacggcaccg aagagctcct cgtgaaactg aatcgggagg atctgctgcg
1981 gaagcagcgc actttcgaca atgggagcat tccccaccag atccatcttg gggagcttca
2041 cgccatcctt cggcgccaag aggacttcta cccctttctt aaggacaaca gggagaagat
2101 tgagaaaatt ctcactttcc gcatccccta ctacgtggga cccctcgcca gaggaaatag
2161 ccggtttgct tggatgacca gaaagtcaga agaaactatc actccctgga acttcgaaga
2221 ggtggtggac aagggagcca gcgctcagtc attcatcgaa cggatgacta acttcgataa
2281 gaacctcccc aatgagaagg tcctgccgaa acattccctg ctctacgagt actttaccgt
2341 gtacaacgag ctgaccaagg tgaaatatgt caccgaaggg atgaggaagc ccgcattcct
2401 gtcaggcgaa caaaagaagg caattgtgga ccttctgttc aagaccaata gaaaggtgac
2461 cgtgaagcag ctgaaggagg actatttcaa gaaaattgaa tgcttcgact ctgtggagat
2521 tagcggggtc gaagatcggt tcaacgcaag cctgggtacc taccatgatc tgcttaagat
2581 catcaaggac aaggattttc tggacaatga ggagaaagag gacatccttg aggacattgt
2641 cctgactctc actctgttcg aggaccggga aatgatcgag gagaggctta agacctacgc
2701 ccatctgttc gacgataaag tgatgaagca acttaaacgg agaagatata ccggatgggg
2761 acgccttagc cgcaaactca tcaacggaat ccgggacaaa cagagcggaa agaccattct
2821 tgatttcctt aagagcgacg gattcgctaa tcgcaacttc atgcaactta tccatgatga
2881 ttccctgacc tttaaggagg acatccagaa ggcccaagtg tctggacaag gtgactcact
2941 gcacgagcat atcgcaaatc tggctggttc acccgctatt aagaagggta ttctccagac
3001 cgtgaaagtc gtggacgagc tggtcaaggt gatgggtcgc cataaaccag agaacattgt
3061 catcgagatg gccagggaaa accagactac ccagaaggga cagaagaaca gcagggagcg
3121 gatgaaaaga attgaggaag ggattaagga gctcgggtca cagatcctta aagagcaccc
3181 ggtggaaaac acccagcttc agaatgagaa gctctatctg tactaccttc aaaatggacg
3241 cgatatgtat gtggaccaag agcttgatat caacaggctc tcagactacg acgtggacgc
3301 catcgtccct cagagcttcc tcaaagacga ctcaattgac aataaggtgc tgactcgctc
3361 agacaagaac cggggaaagt cagataacgt gccctcagag gaagtcgtga aaaagatgaa
3421 gaactattgg cgccagcttc tgaacgcaaa gctgatcact cagcggaagt tcgacaatct
3481 cactaaggct gagaggggcg gactgagcga actggacaaa gcaggattca ttaaacggca
3541 acttgtggag actcggcaga ttactaaaca tgtagcccaa atccttgact cacgcatgaa
3601 taccaagtac gacgaaaacg acaaacttat ccgcgaggtg aaggtgatta ccctgaagtc
3661 caagctggtc agcgatttca gaaaggactt tcaattctac aaagtgcggg agatcaataa
3721 ctatcatcat gctcatgacg catatctgaa tgccgtggtg ggaaccgccc tgatcaagaa
3781 gtacccaaag ctggaaagcg agttcgtgta cggagactac aaggtctacg acgtgcgcaa
3841 gatgattgcc aaatctgagc aggagatcgg aaaggccacc gcaaagtact tcttctacag
3901 caacatcatg aatttcttca agaccgaaat cacccttgca aacggtgaga tccggaagag
3961 gccgctcatc gagactaatg gggagactgg cgaaatcgtg tgggacaagg gcagagattt
4021 cgctaccgtg cgcaaagtgc tttctatgcc tcaagtgaac atcgtgaaga aaaccgaggt
4081 gcaaaccgga ggcttttcta aggaatcaat cctccccaag cgcaactccg acaagctcat
4141 tgcaaggaag aaggattggg accctaagaa gtacggcgga ttcgattcac caactgtggc
4201 ttattctgtc ctggtcgtgg ctaaggtgga aaaaggaaag tctaagaagc tcaagagcgt
4261 gaaggaactg ctgggtatca ccattatgga gcgcagctcc ttcgagaaga acccaattga
4321 ctttctcgaa gccaaaggtt acaaggaagt caagaaggac cttatcatca agctcccaaa
4381 gtatagcctg ttcgaactgg agaatgggcg gaagcggatg ctcgcctccg ctggcgaact
4441 tcagaagggt aatgagctgg ctctcccctc caagtacgtg aatttcctct accttgcaag
4501 ccattacgag aagctgaagg ggagccccga ggacaacgag caaaagcaac tgtttgtgga
4561 gcagcataag cattatctgg acgagatcat tgagcagatt tccgagtttt ctaaacgcgt
4621 cattctcgct gatgccaacc tcgataaagt ccttagcgca tacaataagc acagagacaa
4681 accaattcgg gagcaggctg agaatatcat ccacctgttc accctcacca atcttggtgc
4741 ccctgccgca ttcaagtact tcgacaccac catcgaccgg aaacgctata cctccaccaa
4801 agaagtgctg gacgccaccc tcatccacca gagcatcacc ggactttacg aaactcggat
4861 tgacctctca cagctcggag gggatcccaa gaaaaagcgc aaggtaggat ccatggaacg
4921 tgtgagaatg attaatgtgc aaaggctgtt agaagccgca gagtttttag aaagaagaga
4981 aagagaatgc gaacacgggt atgccagttc tttccctagc atgccctctc ccagaccgaa
5041 aaagaaacgc aaagtttaac tcgagtggca taaagcaatc ttgatgagga taatgatttt
5101 tttttgaata tacataaata ctaccgtttt tctgctagat tttgtgatga cgtaaataag
5161 tacatattac tttttaagcc aagacaagat taagcattaa ctttaccctt ttctttctaa
5221 gtttcaatat tagttatcac tgtttaaaag ttatggcgag aacgtcggcg gttaaaatat
5281 attaccctga acggctgcca aaccagatgt caacacagct acaacgatgt gcttcagtat
5341 tacatttttt gccttcaacg ccttgattgt tctatttttg ctaataataa atctatttca
5401 tcggactaaa agtccattag ttgtaagcgg atttagctca gttgggagag cgccagactg
5461 aagaaaaact tcggtcaagt catctggagg tcctgtgttc gatccacaga attcgcagat
5521 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacaccttc gggtggcgaa
5581 tgggactcgc tatcgtcttc tcacgaagac ttactcagtt ttagagctag aaatagcaag
5641 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
5701 attttttgtc actattgtta tgtaaaatgc cacctctgac agtatggaac gcaaacttct
5761 gtctagtgga tagctgagca gttacagaga tgttacgaac cactagtgca ctgcagtaca
5821 gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc
5881 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
5941 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
6001 cgaagcaaaa attacggctc ctcgctccag acctgcgagc agggaaacgc tcccctcaca
6061 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
6121 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
6181 atcacatccg aacataaaca aaaatgggta aggaaaagac tcacgtttcg aggccgcgat
6241 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
6301 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
6361 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
6421 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
6481 ggttactcac cactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
6541 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
6601 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctggctcag gcgcaatcac
6661 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
6721 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
6781 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
6841 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
6901 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
6961 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taaagtaact
7021 gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta
7081 gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc
7141 tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg
7201 ctggtcgcta tactggagta tcacgtgcta taaaaataat tataatttaa attttttaat
7261 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
7321 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc
7381 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaaaaccaca
7441 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
7501 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
7561 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
7621 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
7681 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtcccg agcggccgcg
7741 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
7801 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
7861 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
7921 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
7981 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
8041 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
8101 accaaaccgt tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta
8161 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
8221 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
8281 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
8341 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
8401 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
8461 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
8521 gcatccatgt tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc
8581 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
8641 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata
8701 aatcgaactt ttgctgagtt gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt
8761 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
8821 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
8881 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
8941 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
9001 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
9061 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
9121 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
9181 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
9241 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
9301 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
9361 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
9421 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
9481 ctgtggataa ccgtgcggcc gc
//
