pThermoCas9_ctrl

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联系方式:I47-825O-882O

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载体基本信息

质粒名称: pThermoCas9_ctrl 
出品公司: 爱迪基因
目录编号: 100981
存储实验室:
John van der Oost

载体骨架基本信息
载体名称: pNW33n
载体抗性: 氯霉素
空载体大小: 4217 bp
完整质粒大小: 7467 bp
载体修饰: 从主干移除810nt长的冗余碎片
载体类型: 细菌表达
生长温度: 37 ℃
宿主菌: DH5a
生长条件: LB培养基+25ug/ml 氯霉素

插入基因1信息
插入基因名称: 来自Geobacillus thermodenitrificans T12的Cas9蛋白
基因又名 ThermoCas9
物种来源: 热反硝化土芽孢杆菌t12 Geobacillus thermodenitrificans T12
插入大小: 3249bp
GenBank ID ARP41326.1
启动子 B. smithii xylL promoter
标签/融合蛋白:
克隆方法: Gibson克隆方法
5' 克隆位点: ATGAAGTATAAAATCGGTCTTG
3' 克隆位点: TTATAACGGACGGATAGTTTC
 
插入基因2信息
插入基因名称 ThermoCas9 single guide RNA expressing module
基因又名 sgRNA
物种 嗜热脱氮芽孢杆菌 T12   Geobacillus thermodenitrificans T12
插入大小 192
启动子 B. coagulans DSM 1 pta promoter
标签
克隆方法 Gibson克隆
5'测序引物: ggatcccatgacgctagtatccagctggGTCATAGTTCCCCTGAGAT
3'测序引物: CCCTCCCATGCACAATAG

文献引用方法
材料和方法部分: pThermoCas9_ctrl was a gift from John van der Oost (Addgene plasmid # 100981 ; http://n2t.net/addgene:100981 ; RRID:Addgene_100981)
参考文献部分: Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der Oost J. Nat Commun. 2017 Nov 21;8(1):1647. doi: 10.1038/s41467-017-01591-4. 10.1038/s41467-017-01591-4 [pii] PubMed 29162801

Gibson克隆方法

载体质粒图谱和多克隆位点信息

pThermoCas9_ctrl质粒图谱

载体简介

载体序列

LOCUS       pThermoCas9_ctrl          7467 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expresses ThermoCas9 and its sgRNA module.
ACCESSION   .
VERSION     .
KEYWORDS    pThermoCas9_ctrl
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7467)
  AUTHORS   Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, 
            Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der 
            Oost J
  TITLE     Characterizing a thermostable Cas9 for bacterial genome editing and 
            silencing.
  JOURNAL   Nat Commun. 2017 Nov 21;8(1):1647. doi: 10.1038/s41467-017-01591-4.
  PUBMED    29162801
REFERENCE   2  (bases 1 to 7467)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7467
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(46..62)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(46..62)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(59..81)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    70..86
                     /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(94..124)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    139..160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1047..2051)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     primer_bind     complement(2572..2589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2743..3331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2823..2842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 actataaaac tattgtgcat gggagggaag cttggcgtaa tcatggtcat agctgtttcc
       61 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
      121 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
      181 cgctttccag tcgggaaacc tgtcgtgcca gcccttcaaa cttcccaaag gcgagcccta
      241 gtgacattag aaaaccgact gtaaaaagta cagtcggcat tatctcatat tataaaagcc
      301 agtcattagg cctatctgac aattcctgaa tagagttcat aaacaatcct gcatgataac
      361 catcacaaac agaatgatgt acctgtaaag atagcggtaa atatattgaa ttacctttat
      421 taatgaattt tcctgctgta ataatgggta gaaggtaatt actattatta ttgatattta
      481 agttaaaccc agtaaatgaa gtccatggaa taatagaaag agaaaaagca ttttcaggta
      541 taggtgtttt gggaaacaat ttccccgaac cattatattt ctctacatca gaaaggtata
      601 aatcataaaa ctctttgaag tcattcttta caggagtcca aataccagag aatgttttag
      661 atacaccatc aaaaattgta taaagtggct ctaacttatc ccaataacct aactctccgt
      721 cgctattgta accagttcta aaagctgtat ttgagtttat cacccttgtc actaagaaaa
      781 taaatgcagg gtaaaattta tatccttctt gttttatgtt tcggtataaa acactaatat
      841 caatttctgt ggttatacta aaagtcgttt gttggttcaa ataatgatta aatatctctt
      901 ttctcttcca attgtctaaa tcaattttat taaagttcat ttgatatgcc tcctaaattt
      961 ttatctaaag tgaatttagg aggcttactt gtctgctttc ttcattagaa tcaatccttt
     1021 tttaaaagtc aatcccgttt gttgaactac tctttaataa aataattttt ccgttcccaa
     1081 ttccacattg caataataga aaatccatct tcatcggctt tttcgtcatc atctgtatga
     1141 atcaaatcgc cttcttctgt gtcatcaagg tttaattttt tatgtatttc ttttaacaaa
     1201 ccaccatagg agattaacct tttacggtgt aaaccttcct ccaaatcaga caaacgtttc
     1261 aaattctttt cttcatcatc ggtcataaaa tccgtatcct ttacaggata ttttgcagtt
     1321 tcgtcaattg ccgattgtat atccgattta tatttatttt tcggtcgaat catttgaact
     1381 tttacatttg gatcatagtc taatttcatt gcctttttcc aaaattgaat ccattgtttt
     1441 tgattcacgt agttttctgt attcttaaaa taagttggtt ccacacatac caatacatgc
     1501 atgtgctgat tataagaatt atctttatta tttattgtca cttccgttgc acgcataaaa
     1561 ccaacaagat ttttattaat ttttttatat tgcatcattc ggcgaaatcc ttgagccata
     1621 tctgacaaac tcttatttaa ttcttcgcca tcataaacat ttttaactgt taatgtgaga
     1681 aacaaccaac gaactgttgg cttttgttta ataacttcag caacaacctt ttgtgactga
     1741 atgccatgtt tcattgctct cctccagttg cacattggac aaagcctgga tttacaaaac
     1801 cacactcgat acaactttct ttcgcctgtt tcacgatttt gtttatactc taatatttca
     1861 gcacaatctt ttactctttc agccttttta aattcaagaa tatgcagaag ttcaaagtaa
     1921 tcaacattag cgattttctt ttctctccat ggtctcactt ttccactttt tgtcttgtcc
     1981 actaaaaccc ttgatttttc atctgaataa atgctactat taggacacat aatattaaaa
     2041 gaaaccccca tctatttagt tatttgtttg gtcacttata actttaacag atggggtttt
     2101 tctgtgcaac caattttaag ggttttcaat actttaaaac acatacatac caacacttca
     2161 acgcaccttt cagcaactaa aataaaaatg acgttatttc tatatgtatc aagaatagaa
     2221 agaactcgtt tttcgctacg ctcaaaacgc aaaaaaagca ctcattcgag tgctttttct
     2281 tatcgctcca aatcatgcga ttttttcctc tttgcttttc tttgctcacg aagttctcga
     2341 tcacgctgca aaacatcttg aagcgaaaaa gtattcttct tttcttccga tcgctcatgc
     2401 tgacgcacga aaagccctct aggcgcatag gaacaactcc taaatgcatg tgaggggttt
     2461 tctcgtccat gtgaacagtc gcatacgcaa tattttgttt cccatactgc attaatgaat
     2521 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2581 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2641 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2701 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2761 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2821 ataaagatac caggcgtttc cccatggaag ctccctcgtg cgctctcctg ttccgaccct
     2881 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2941 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3001 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3061 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3121 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3181 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3241 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3301 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3361 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagaa catcctcttt
     3421 cttagtttgt atagtcaata aacgaactaa attttcattt ttatcttttt ctagacattt
     3481 ttattgtatc gtatttttta ttcaaaaaaa atattgaatt ttaaaaatga tggtgcagta
     3541 tgaaggtgta cttagtttat tggttaaaca aagtacgcat caatatattt tttttgatgc
     3601 gatttgaatt cgctttcatt atctaaaaca ttcaatcgag gaggtaactt aaatgaagta
     3661 taaaatcggt cttgatatcg gcattacgtc tatcggttgg gctgtcatta atttggacat
     3721 tcctcgcatc gaagatttag gtgtccgcat ttttgacaga gcggaaaacc cgaaaaccgg
     3781 ggagtcacta gctcttccac gtcgcctcgc ccgctccgcc cgacgtcgtc tgcggcgtcg
     3841 caaacatcga ctggagcgca ttcgccgcct gttcgtccgc gaaggaattt taacgaagga
     3901 agagctgaac aagctgtttg aaaaaaagca cgaaatcgac gtctggcagc ttcgtgttga
     3961 agcactggat cgaaaactaa ataacgatga attagcccgc atccttcttc atctggctaa
     4021 acggcgtgga tttagatcca accgcaagag tgagcgcacc aacaaagaaa acagtacgat
     4081 gctcaaacat attgaagaaa accaatccat tctttcaagt taccgaacgg ttgcagaaat
     4141 ggttgtcaag gatccgaaat tttccctgca caagcgtaat aaagaggata attacaccaa
     4201 cactgttgcc cgcgacgatc ttgaacggga aatcaaactg attttcgcca aacagcgcga
     4261 atatgggaac atcgtttgca cagaagcatt tgaacacgag tatatttcca tttgggcatc
     4321 gcaacgccct tttgcttcta aggatgatat cgagaaaaaa gtcggtttct gtacgtttga
     4381 gcctaaagaa aaacgcgcgc caaaagcaac atacacattc cagtccttca ccgtctggga
     4441 acatattaac aaacttcgtc ttgtctcccc gggaggcatc cgggcactaa ccgatgatga
     4501 acgtcgtctt atatacaagc aagcatttca taaaaataaa atcaccttcc atgatgttcg
     4561 aacattgctt aacttgcctg acgacacccg ttttaaaggt cttttatatg accgaaacac
     4621 cacgctgaag gaaaatgaga aagttcgctt ccttgaactc ggcgcctatc ataaaatacg
     4681 gaaagcgatc gacagcgtct atggcaaagg agcagcaaaa tcatttcgtc cgattgattt
     4741 tgatacattt ggctacgcat taacgatgtt taaagacgac accgacattc gcagttactt
     4801 gcgaaacgaa tacgaacaaa atggaaaacg aatggaaaat ctagcggata aagtctatga
     4861 tgaagaattg attgaagaac ttttaaactt atcgttttct aagtttggtc atctatccct
     4921 taaagcgctt cgcaacatcc ttccatatat ggaacaaggc gaagtctact caaccgcttg
     4981 tgaacgagca ggatatacat ttacagggcc aaagaaaaaa cagaaaacgg tattgctgcc
     5041 gaacattccg ccgatcgcca atccggtcgt catgcgcgca ctgacacagg cacgcaaagt
     5101 ggtcaatgcc attatcaaaa agtacggctc accggtctcc atccatatcg aactggcccg
     5161 ggaactatca caatcctttg atgaacgacg taaaatgcag aaagaacagg aaggaaaccg
     5221 aaagaaaaac gaaactgcca ttcgccaact tgttgaatat gggctgacgc tcaatccaac
     5281 tgggcttgac attgtgaaat tcaaactatg gagcgaacaa aacggaaaat gtgcctattc
     5341 actccaaccg atcgaaatcg agcggttgct cgaaccaggc tatacagaag tcgaccatgt
     5401 gattccatac agccgaagct tggacgatag ctataccaat aaagttcttg tgttgacaaa
     5461 ggagaaccgt gaaaaaggaa accgcacccc agctgaatat ttaggattag gctcagaacg
     5521 ttggcaacag ttcgagacgt ttgtcttgac aaataagcag ttttcgaaaa agaagcggga
     5581 tcgactcctt cggcttcatt acgatgaaaa cgaagaaaat gagtttaaaa atcgtaatct
     5641 aaatgatacc cgttatatct cacgcttctt ggctaacttt attcgcgaac atctcaaatt
     5701 cgccgacagc gatgacaaac aaaaagtata cacggtcaac ggccgtatta ccgcccattt
     5761 acgcagccgt tggaatttta acaaaaaccg ggaagaatcg aatttgcatc atgccgtcga
     5821 tgctgccatc gtcgcctgca caacgccgag cgatatcgcc cgagtcaccg ccttctatca
     5881 acggcgcgaa caaaacaaag aactgtccaa aaagacggat ccgcagtttc cgcagccttg
     5941 gccgcacttt gctgatgaac tgcaggcgcg tttatcaaaa aatccaaagg agagtataaa
     6001 agctctcaat cttggaaatt atgataacga gaaactcgaa tcgttgcagc cggtttttgt
     6061 ctcccgaatg ccgaagcgga gcataacagg agcggctcat caagaaacat tgcggcgtta
     6121 tatcggcatc gacgaacgga gcggaaaaat acagacggtc gtcaaaaaga aactatccga
     6181 gatccaactg gataaaacag gtcatttccc aatgtacggg aaagaaagcg atccaaggac
     6241 atatgaagcc attcgccaac ggttgcttga acataacaat gacccaaaaa aggcgtttca
     6301 agagcctctg tataaaccga agaagaacgg agaactaggt cctatcatcc gaacaatcaa
     6361 aatcatcgat acgacaaatc aagttattcc gctcaacgat ggcaaaacag tcgcctacaa
     6421 cagcaacatc gtgcgggtcg acgtctttga gaaagatggc aaatattatt gtgtccctat
     6481 ctatacaata gatatgatga aagggatctt gccaaacaag gcgatcgagc cgaacaaacc
     6541 gtactctgag tggaaggaaa tgacggagga ctatacattc cgattcagtc tatacccaaa
     6601 tgatcttatc cgtatcgaat ttccccgaga aaaaacaata aagactgctg tgggggaaga
     6661 aatcaaaatt aaggatctgt tcgcctatta tcaaaccatc gactcctcca atggagggtt
     6721 aagtttggtt agccatgata acaacttttc gctccgcagc atcggttcaa gaaccctcaa
     6781 acgattcgag aaataccaag tagatgtgct aggcaacatc tacaaagtga gaggggaaaa
     6841 gagagttggg gtggcgtcat cttctcattc gaaagccggg gaaactatcc gtccgttata
     6901 aatcagacaa aatggcctgc ttatgaagcg ggccattttt gtttaatcct gtcgacttgc
     6961 cggaattctt tcacagccgc atccctttct tccgcaggca gcgccttcca gcccgcccag
     7021 tcgagagaac ggaaatcatg cagtgagtac cagccatcta gtgtttctgc tgcttcgctc
     7081 atttttctcc ctccaaatgt ttttctcttc ccttactata acacagttta gaagagggag
     7141 aaaatattac ggctgccgcg aaattgtcta aaagaagcct tcttttgcct ttgattttcc
     7201 ggaaaaatgg attattctta aaatggatac cgttatacgg atcccatgac gctagtatcc
     7261 agctgggtca tagttcccct gagattatcg ctgtggtata atgaaagtta taccacagca
     7321 atgatctcag ggttactatg ataagggctt tctgcctata ggcagactga cccgtggcgt
     7381 tggggatcgc ctatcgcccg ctttcttcgg gcattcccca ctcttaggcg aacgaagacc
     7441 ttggcggaaa acgtcaaggt ctttttt
//

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