| 质粒名称: | His6-pAG-RTase |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 214932 |
| 存储实验室: | Chuan He |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Clontech |
| 空载体大小: | 5369bp |
| 完整质粒大小: | 7495bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | pAG-RTase、fusion protein of protein A/G and MMLV reverse transcriptase (25-497) |
| 插入大小: | 2157bp |
| 基因突变: | MMLV RTase (H8Y, D200N, T306K, W313F, T330P, D524G, L603W) |
| 启动子: | T7 promoter |
| 标签/融合蛋白: | His6 (N terminal on insert) |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | T7F |
| 3’克隆位点: | T7R |
|
文献引用方法 |
|
| 材料和方法部分: | His6-pAG-RTase was a gift from Chuan He (Addgene plasmid # 214932 ; http://n2t.net/addgene:214932 ; RRID:Addgene_214932) |
| 参考文献部分: | Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing. Xiao Y, Chen YM, Zou Z, Ye C, Dou X, Wu J, Liu C, Liu S, Yan H, Wang P, Zeng TB, Liu Q, Fei J, Tang W, He C. Nat Methods. 2024 Jan 10. doi: 10.1038/s41592-023-02146-w. 10.1038/s41592-023-02146-w PubMed 38200227 |
买家导航
His6-pAG-RTase载体质粒基本信息
His6-pAG-RTase质粒图谱载体图谱和His6-pAG-RTase载体序列质粒序列多克隆位点信息
His6-pAG-RTase质粒载体简介
His6-pAG-RTase质粒序列载体序列
LOCUS His6-pAG-RTase 7491 bp DNA circular SYN 14-APR-2025
DEFINITION Express fusion proteins of Protein A/G and MMLV reverse
transcriptase (25-497) in bacterial systems.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7491)
AUTHORS Xiao Y, Chen YM, Zou Z, Ye C, Dou X, Wu J, Liu C, Liu S, Yan H, Wang
P, Zeng TB, Liu Q, Fei J, Tang W, He C
TITLE Profiling of RNA-binding protein binding sites by in situ reverse
transcription-based sequencing.
JOURNAL Nat Methods. 2024 Jan 10. doi: 10.1038/s41592-023-02146-w.
PUBMED 38200227
REFERENCE 2 (bases 1 to 7491)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2024 Jan 10. doi: 10.1038/s41592-023-02146-w."
COMMENT Sequence Label: His6-pAG-RTase
FEATURES Location/Qualifiers
source 1..7491
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 316..525
/codon_start=1
/product="immunoglobulin binding domain of Streptoccocus
protein G"
/label=protein G
/translation="TTYKLVINGKTLKGETTTEAVDAETAERHFKQYANDNGVDGEWTY
DDATKTFTVTEKPEVIDASELTPAV"
CDS 2044..2061
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2114..2132)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2128..2175
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2212..2667
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2299..2318)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2509..2530
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2759..3574)
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
CDS complement(2759..3574)
/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 3482..3501
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3566..3585
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 3696..4284
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4185..4204
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4438..4455
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4470..4609
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4535..4554)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4695..4717
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4711..4902)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5711..6793)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 6755..6774
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6794..6871)
/gene="lacI"
/label=lacI promoter
primer_bind 7077..7096
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 7180..7199
/label=T7
/note="T7 promoter, forward primer"
promoter 7180..7198
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 7199..7223
/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)."
RBS 7238..7260
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 7279..7296
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 7306..7323
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 7327..7359
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/label=T7 tag (gene 10 leader)
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS join(7381..7491,1..315)
/codon_start=1
/product="immunoglobulin binding domains of Staphylocuccus
aureus protein A"
/label=protein A
/translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
ORIGIN
1 gctttctatg aaatcttaca tttacctaac ttaaacgaag aacaacgcaa tggtttcatc
61 caaagcctaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc taaaaagcta
121 aatgatgctc aagcaccaaa agctgacaac aaattcaaca aagaacaaca aaatgctttc
181 tatgaaattt tacatttacc taacttaact gaagaacaac gtaacggctt catccaaagc
241 cttaaagacg atccttcagt gagcaaagaa attttagcag aagctaaaaa gctaaacgat
301 gctcaagcac caaaaacaac ttataaatta gtcatcaacg ggaaaacgct gaagggtgaa
361 accacgacag aggccgtaga tgcggagaca gcggagcgcc actttaagca atacgcgaat
421 gataacggtg tagacggcga gtggacctac gacgacgcga caaagacctt taccgtcacg
481 gagaaacctg aggttatcga cgcgtctgag ttgacgccag ccgtatctag cggaggatcc
541 tctggcagcg agacaccagg aacaagcgag tcagcaacac cagagagcag tggcggcagc
601 agcggcggca gcagctggct gtctgatttt cctcaggcct gggcggaaac cgggggcatg
661 ggactggcag ttcgccaagc tcctctgatc atacctctga aagcaacctc tacccccgtg
721 tccataaaac aataccccat gtcacaagaa gccagactgg ggatcaagcc ccacatacag
781 agactgttgg accagggaat actggtaccc tgccagtccc cctggaacac gcccctgcta
841 cccgttaaga aaccagggac taatgattat aggcctgtcc aggatctgag agaagtcaac
901 aagcgggtgg aagacatcca ccccaccgtg cccaaccctt acaacctctt gagcgggctc
961 ccaccgtccc accagtggta cactgtgctt gatttaaagg atgccttttt ctgcctgaga
1021 ctccacccca ccagtcagcc tctcttcgcc tttgagtgga gagatccaga gatgggaatc
1081 tcaggacaat tgacctggac cagactccca cagggtttca aaaacagtcc caccctgttt
1141 aatgaggcac tgcacagaga cctagcagac ttccggatcc agcacccaga cttgatcctg
1201 ctacagtacg tggatgactt actgctggcc gccacttctg agctagactg ccaacaaggt
1261 actcgggccc tgttacaaac cctagggaac ctcgggtatc gggcctcggc caagaaagcc
1321 caaatttgcc agaaacaggt caagtatctg gggtatcttc taaaagaggg tcagagatgg
1381 ctgactgagg ccagaaaaga gactgtgatg gggcagccta ctccgaagac ccctcgacaa
1441 ctaagggagt tcctagggaa ggcaggcttc tgtcgcctct tcatccctgg gtttgcagaa
1501 atggcagccc ccctgtaccc tctcaccaaa ccggggactc tgtttaattg gggcccagac
1561 caacaaaagg cctatcaaga aatcaagcaa gctcttctaa ctgccccagc cctggggttg
1621 ccagatttga ctaagccctt tgaactcttt gtcgacgaga agcagggcta cgccaaaggt
1681 gtcctaacgc aaaaactggg accttggcgt cggccggtgg cctacctgtc caaaaagcta
1741 gacccagtag cagctgggtg gcccccttgc ctacggatgg tagcagccat tgccgtactg
1801 acaaaggatg caggcaagct aaccatggga cagccactag tcattctggc cccccatgca
1861 gtagaggcac tagtcaaaca accccccgac cgctggcttt ccaacgcccg gatgactcac
1921 tatcaggcct tgcttttgga cacggaccgg gtccagttcg gaccggtggt agccctgaac
1981 ccggctacgc tgctcccact gcctgaggaa gggctgcaac acaactgcct tgattaactc
2041 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
2101 ttggctgctg ccgccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
2161 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
2221 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
2281 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
2341 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
2401 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
2461 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
2521 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
2581 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
2641 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
2701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
2761 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
2821 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
2881 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
2941 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
3001 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
3061 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
3121 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
3181 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
3241 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
3301 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
3361 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
3421 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
3481 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
3541 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
3601 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
3661 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
3721 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
3781 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
3841 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
3901 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
3961 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
4021 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
4081 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
4141 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
4201 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
4261 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
4321 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
4381 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
4441 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
4501 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
4561 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
4621 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
4681 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
4741 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
4801 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
4861 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
4921 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
4981 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
5041 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
5101 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
5161 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
5221 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
5281 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
5341 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
5401 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
5461 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
5521 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
5581 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
5641 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
5701 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
5761 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
5821 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
5881 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
5941 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
6001 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
6061 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
6121 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
6181 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
6241 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
6301 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
6361 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
6421 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
6481 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
6541 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
6601 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
6661 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
6721 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
6781 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
6841 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
6901 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
6961 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
7021 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
7081 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
7141 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
7201 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
7261 tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc
7321 agccatatgg ctagcatgac tggtggacag caaatgggtc gcggatccat gattacgcca
7381 agcttaaaag atgacccaag ccaaagtgct aacctattgt cagaagctaa aaagttaaat
7441 gaatctcaag caccgaaagc ggataacaaa ttcaacaaag aacaacaaaa t
//
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His6-pAG-RTase质粒载体应用举例
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