| 出品公司: | Addgene |
|---|---|
| 载体名称: | pHD_v2 |
| 质粒类型: | 靶向基因操作载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 启动子: | -- |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 载体大小: | 2746bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | 卡纳霉素（Kanamycin） |
| 筛选标记: | -- |
| 备注: | -- |
| 产品目录号: | 32183 |
| 稳定性: | -- |
| 组成型: | -- |
| 病毒/非病毒: | -- |
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载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
(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.载体序列
LOCUS pHD_v2 2746 bp DNA SYN
DEFINITION pHD_v2
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..2746
/organism="pHD_v2"
/mol_type="other DNA"
CDS complement(0..809)
/label="ORF frame 3"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
gene complement(12..809)
/label="KanR2 (variant)"
/gene="KanR2 (variant)"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
promoter complement(851..879)
/label="AmpR_promoter"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
terminator 990..1147
/label="rrnB_terminator"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
promoter complement(1748..1764)
/label="M13_forward20_primer"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin complement(2019..2638)
/label="pBR322_origin"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
gene complement(2746..809)
/label="KanR2 (variant)"
/gene="KanR2 (variant)"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
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 TTGCGAGCCA TGACGGGGGA 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
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