| 质粒名称: | 1GFP/RNase H1 D210N |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 174448 |
| 存储实验室: | Karlene Cimprich |
|
载体骨架基本信息 |
|
| 载体名称: | 1GFP |
| 载体出品公司: | 加州大学伯克利分校加州定量生物科学研究所宏观实验室 |
| 空载体大小: | 6075 bp |
| 完整质粒大小: | 6858 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 卡那霉素，50μg/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: | RNase H1 |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 783 bp |
| 基因突变: | D210N |
| GenBank编号 | -- |
| 启动子: | T7 promoter |
| 标签/融合蛋白: | His-tag, GFP (N terminal on backbone) |
| 克隆方法: | Gibson克隆 |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | GFP forward primer: 5'-GTCCTGCTGGAGTTCGTGACC-3' |
| 3’测序引物 | T7 terminator reverse primer |
|
文献引用方法 |
|
| 材料和方法部分: | 1GFP/RNase H1 D210N was a gift from Karlene Cimprich (Addgene plasmid # 174448 ; http://n2t.net/addgene:174448 ; RRID:Addgene_174448) |
| 参考文献部分: | Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging. Crossley MP, Brickner JR, Song C, Zar SMT, Maw SS, Chedin F, Tsai MS, Cimprich KA. J Cell Biol. 2021 Sep 6;220(9). pii: 212458. doi: 10.1083/jcb.202101092. Epub 2021 Jul 7. 10.1083/jcb.202101092 PubMed 34232287 |
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 1GFP/RNase H1 D210N 6855 bp ds-DNA circular SYN 07-SEP-2021
DEFINITION Express GFP-tagged RNase H1 D210N in E. coli..
ACCESSION .
VERSION .
KEYWORDS 1GFP/RNase H1 D210N
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6855)
AUTHORS Crossley MP, Brickner JR, Song C, Zar SMT, Maw SS, Chedin F, Tsai
MS, Cimprich KA
TITLE Catalytically inactive, purified RNase H1: A specific and sensitive
probe for RNA-DNA hybrid imaging.
JOURNAL J Cell Biol. 2021 Sep 6;220(9). pii: 212458. doi:
10.1083/jcb.202101092. Epub 2021 Jul 7.
PUBMED 34232287
REFERENCE 2 (bases 1 to 6855)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 7, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6855
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(696..1778)
/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 1740..1759
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1779..1856)
/gene="lacI"
/label=lacI promoter
primer_bind 2062..2081
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 2165..2184
/label=T7
/note="T7 promoter, forward primer"
promoter 2165..2183
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2184..2208
/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 2223..2245
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2264..2281
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 2291..3004
/codon_start=1
/product="enhanced GFP (EGFP) derivative that exhibits
robust folding and enhanced photostability (Zhong et al.,
2019)"
/label=SiriusGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(2333..2354)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 2941..2962
/label=EGFP-C
/note="EGFP, forward primer"
CDS 3014..3034
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 3883..3900
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3953..3971)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3967..4014
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 4051..4506
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4138..4157)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4348..4369
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(4599..5414)
/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 5322..5341
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 5406..5425
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 5536..6124
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6025..6044
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6278..6295
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 6310..6449
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(6375..6394)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6535..6557
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(6551..6742)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
61 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
121 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
181 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
241 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
301 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
361 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
421 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
481 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
541 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
601 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
661 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
721 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
781 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
841 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
901 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
961 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
1021 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc
1081 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
1141 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
1201 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
1261 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
1321 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
1381 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
1441 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
1501 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
1561 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
1621 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
1681 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
1741 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
1801 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
1861 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
1921 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
1981 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
2041 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
2101 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
2161 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
2221 attttgttta actttaagaa ggagatatac catgggttct tctcaccatc accatcacca
2281 tggttcttct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
2341 gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc
2401 caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
2461 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
2521 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
2581 catctccttc aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga
2641 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
2701 ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca
2761 gaagaacggc atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca
2821 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
2881 caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca
2941 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
3001 caaggggatc gaggaaaacc tgtacttcca atccaatatg ttctatgccg tgaggagggg
3061 ccgcaagacc ggggtctttc tgacctggaa tgagtgcaga gcacaggtgg accggtttcc
3121 tgctgccaga tttaagaagt ttgccacaga ggatgaggcc tgggcctttg tcaggaaatc
3181 tgcaagcccg gaagtttcag aagggcatga aaatcaacat ggacaagaat cggaggcgaa
3241 agccagcaag cgactccgtg agccactgga tggagatgga catgaaagcg cagagccgta
3301 tgcaaagcac atgaagccga gcgtggagcc ggcgcctcca gttagcagag acacgttttc
3361 ctacatggga gacttcgtcg tcgtctacac tgatggctgc tgctccagta atgggcgtag
3421 aaggccgcga gcaggaatcg gcgtttactg ggggccgggc catcctttaa atgtaggcat
3481 tagacttcct gggcggcaga caaaccaaag agcggaaatt catgcagcct gcaaagccat
3541 tgaacaagca aagactcaaa acatcaataa actggttctg tatacaaaca gtatgtttac
3601 gataaatggt ataactaact gggttcaagg ttggaagaaa aatgggtgga agacaagtgc
3661 agggaaagag gtgatcaaca aagaggactt tgtggcactg gagaggctta cccaggggat
3721 ggacattcag tggatgcatg ttcctggtca ttcgggattt ataggcaatg aagaagctga
3781 cagattagcc agagaaggag ctaaacaatc ggaagactaa attggaagtg gataacggat
3841 ccgaattcga gcgccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac
3901 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
3961 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
4021 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
4081 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
4141 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
4201 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
4261 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
4321 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
4381 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
4441 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaacta gtaacgttta
4501 caatttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
4561 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
4621 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
4681 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
4741 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
4801 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
4861 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
4921 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
4981 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
5041 gccagcgcat caacaatgtt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
5101 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
5161 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
5221 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
5281 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
5341 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
5401 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
5461 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
5521 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
5581 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
5641 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
5701 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
5761 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
5821 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
5881 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
5941 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
6001 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
6061 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
6121 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
6181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
6241 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
6301 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
6361 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
6421 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
6481 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
6541 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
6601 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
6661 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
6721 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
6781 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
6841 cccggttact ggaac
//
载体图谱质粒图谱pdf版和相关资料下载
载体质粒应用举例
- 上一篇：pET28a-uvsX-H6
- 下一篇：pSecUAG-Evol2
