產品名稱：pNN_v2靶向基因操作載體

基本信息

質粒圖譜

載體序列

LOCUS       pNN_v2	2746 bp 	DNA	SYN
DEFINITION  pNN_v2
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..2746
                     /organism="pNN_v2"
                     /mol_type="other DNA"
     CDS             complement(0..809)
                     /label="ORF frame 3"
                   
     gene            complement(12..809)
                     /label="KanR2 (variant)"
                     /gene="KanR2 (variant)"
                   
     promoter        complement(851..879)
                     /label="AmpR_promoter"
                     
     terminator      990..1147
                     /label="rrnB_terminator"
                    
     promoter        complement(1748..1764)
                     /label="M13_forward20_primer"
                    
     rep_origin      complement(2019..2638)
                     /label="pBR322_origin"
           
     gene            complement(2746..809)
                     /label="KanR2 (variant)"
                     /gene="KanR2 (variant)"
     
ORIGIN
    1 TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA
   61 CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT
  121 AGGATGGCAA GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT
  181 ATTAATTTCC CCTCGTCAAA AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT
  241 GAATCCGGTG AGAATGGCAA AAGTTTATGC ATTTCTTTCC AGACTTGTTC AACAGGCCAG
  301 CCATTACGCT CGTCATCAAA ATCACTCGCA TCAACCAAAC CGTTATTCAT TCGTGATTGC
  361 GCCTGAGCGA GGCGAAATAC GCGATCGCTG TTAAAAGGAC AATTACAAAC AGGAATCGAG
  421 TGCAACCGGC GCAGGAACAC TGCCAGCGCA TCAACAATAT TTTCACCTGA ATCAGGATAT
  481 TCTTCTAATA CCTGGAACGC TGTTTTTCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA
  541 TCAGGAGTAC GGATAAAATG CTTGATGGTC GGAAGTGGCA TAAATTCCGT CAGCCAGTTT
  601 AGTCTGACCA TCTCATCTGT AACATCATTG GCAACGCTAC CTTTGCCATG TTTCAGAAAC
  661 AACTCTGGCG CATCGGGCTT CCCATACAAG CGATAGATTG TCGCACCTGA TTGCCCGACA
  721 TTATCGCGAG CCCATTTATA CCCATATAAA TCAGCATCCA TGTTGGAATT TAATCGCGGC
  781 CTCGACGTTT CCCGTTGAAT ATGGCTCATA TTCTTCCTTT TTCAATATTA TTGAAGCATT
  841 TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA
  901 ATAGGGGTCA GTGTTACAAC CAATTAACCA ATTCTGAACA TTATCGCGAG CCCATTTATA
  961 CCTGAATATG GCTCATAACA CCCCTTGTTT GCCTGGCGGC AGTAGCGCGG TGGTCCCACC
 1021 TGACCCCATG CCGAACTCAG AAGTGAAACG CCGTAGCGCC GATGGTAGTG TGGGGACTCC
 1081 CCATGCGAGA GTAGGGAACT GCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT
 1141 GGGCCTTTCG CCCGGGCTAA TTAGGGGGTG TCGCCCTTCG CTGAACTCAC CCCAGAGCAG
 1201 GTCGTGGCAA TTGCGAGCAA CAACGGGGGA AAGCAGGCAC TCGAAACCGT CCAGAGGTTG
 1261 CTGCCTGTGC TGTGCCAAGC GCACGGACGT CAAAAGGGCG ACACAAAATT TATTCTAAAT
 1321 GCATAATAAA TACTGATAAC ATCTTATAGT TTGTATTATA TTTTGTATTA TCGTTGACAT
 1381 GTATAATTTT GATATCAAAA ACTGATTTTC CCTTTATTAT TTTCGAGATT TATTTTCTTA
 1441 ATTCTCTTTA ACAAACTAGA AATATTGTAT ATACAAAAAA TCATAAATAA TAGATGAATA
 1501 GTTTAATTAT AGGTGTTCAT CAATCGAAAA AGCAACGTAT CTTATTTAAA GTGCGTTGCT
 1561 TTTTTCTCAT TTATAAGGTT AAATAATTCT CATATATCAA GCAAAGTGAC AGGCGCCCTT
 1621 AAATATTCTG ACAAATGCTC TTTCCCTAAA CTCCCCCCAT AAAAAAACCC GCCGAAGCGG
 1681 GTTTTTACGT TATTTGCGGA TTAACGATTA CTCGTTATCA GAACCGCCCA GGGGGCCCGA
 1741 GCTTAAGACT GGCCGTCGTT TTACAACACA GAAAGAGTTT GTAGAAACGC AAAAAGGCCA
 1801 TCCGTCAGGG GCCTTCTGCT TAGTTTGATG CCTGGCAGTT CCCTACTCTC GCCTTCCGCT
 1861 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC
 1921 TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA
 1981 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT
 2041 AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC
 2101 CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT
 2161 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG
 2221 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG
 2281 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT
 2341 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG
 2401 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GGCTAACTAC
 2461 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA
 2521 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT
 2581 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT
 2641 TCTACGGGGT CTGACGCTCA GTGGAACGAC GCGCGCGTAA CTCACGTTAA GGGATTTTGG
 2701 TCATGAGCTT GCGCCGTCCC GTCAAGTCAG CGTAATGCTC TGCTTT

載體描述

(a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA-binding module consists of 34 amino acids, where the  RVDs in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA-binding modules are flanked by nonrepetitive N and C termini, which carry the  translocation, nuclear localization (NLS) and transcription activation (AD) domains. A cryptic signal within the N terminus  specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids  in total: four monomer plasmids to be used as templates for PCR amplification, four TALE-TF and four TALEN cloning backbones  corresponding to four different bases targeted by the 0.5 repeat. CMV, cytomegalovirus promoter; N term, nonrepetitive N  terminus from the Hax3 TALE; C term, nonrepetitive C terminus from the Hax3 TALE; BsaI, type IIs restriction sites used for  the insertion of custom TALE DNA-binding domains; ccdB + CmR, negative selection cassette containing the ccdB negative  selection gene and chloramphenicol resistance gene; NLS, nuclear localization signal; VP64, synthetic transcriptional  activator derived from VP16 protein of herpes simplex virus; 2A, 2A self-cleavage linker; EGFP, enhanced green fluorescent  protein; polyA signal, polyadenylation signal; FokI, catalytic domain from the FokI endonuclease.  (c) TALEs can be used to generate custom TALE-TFs and modulate the transcription of endogenous genes from the genome. The  TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription.  (d) TALENs can be used to generate site-specific double-strand breaks to facilitate genome editing through nonhomologous  repair or homology directed repair. Two TALENs target a pair of binding sites flanking a 16-bp spacer. The left and right  TALENs recognize the top and bottom strands of the target sites, respectively. Each TALE DNA-binding domain is fused to the  catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right  TALEN-binding sites.

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