| 出品公司: | Invitrogen |
|---|---|
| 载体名称: | pET100 ； pET100/D-TOPO |
| 质粒类型: | 大肠杆菌表达载体；细菌表达载体；TOPO载体 |
| 高拷贝/低拷贝: | 低拷贝 |
| 克隆方法: | TOPO |
| 启动子: | T7 |
| 载体大小: | 5764 bp |
| 5' 测序引物及序列: | T7 promoter 5’TAATACGACTCACTATAGGG 3’ |
| 3' 测序引物及序列: | T7 terminator 5’GCTAGTTATTGCTCAGCGG 3’ |
| 载体标签: | 6xHis tag、Xpress tag（N-term）；T7 tag （C-term） |
| 载体抗性: | 氨苄青霉素 |
| 克隆菌株: | TOP10 |
| 表达菌株: | BL21-Star(DE3) 、BL21-Star(DE3) pLysS |
| 备注: | pET100含有T7聚合酶结合位点和RBS核糖体结合位点，可以做体外转录和翻译。 |
| 产品目录号: | ZY100-01 |
| 组成型/诱导型: | 诱导型 |
| 病毒/非病毒: | 非病毒 |
- ¥2500
- ZYbscience
- 中国/美国
- ZY100-01
- 2025年07月12日
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pET100/D-TOPO
- 库存：
20
- 供应商：
泽叶生物
pET100载体基本信息
pET100载体质粒图谱和多克隆位点信息
pET100载体简介
pET100载体序列
LOCUS pET100/D-TOPO 5764 bp ds-DNA circular SYN 15-AUG-2016
DEFINITION synthetic circular DNA
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5764)
AUTHORS Biofeng Lab.
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5764
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 209..227
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 228..252
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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 282..288
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
misc_feature 297..299
/note="ATG"
/note="Initiation ATG:"
CDS 309..326
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS 330..362
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10
T7 tag (gene 10 leader)"
/note="T7 tag"
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS 366..389
/codon_start=1
/product="Xpress(TM) epitope tag, including an enterokinase
recognition and cleavage site"
/note="Xpress(TM) tag"
/translation="DLYDDDDK"
misc_feature 396..400
/note="TOPO site1"
/note="TOPO recognition site1"
misc_feature 405..409
/note="TOPO site 2"
/note="TOPO recognition site 2"
terminator 427..555
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(705..733)
/note="tet promoter"
/note="E. coli promoter for tetracycline efflux protein
gene"
promoter 856..954
/gene="bla"
/note="bla promoter"
CDS 955..1815
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2022..2757
/direction=RIGHT
/note="pBR322"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 2760..2899
/note="bom"
/note="basis of mobility region from pBR322"
CDS complement(3001..3192)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(4507..5595)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
SPRALADSLMQLARQVSRLESGQ"
promoter complement(5599..5664)
/gene="lacI"
/note="lacI promoter"
ORIGIN
1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
301 ggggttctca tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg
361 gtcgggatct gtacgacgat gacgataagg atcatccctt caccaagggc gagctcaacg
421 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
481 aactagcata accccttggg gcctctaaac gggtcttgag gagttttttg ctgaaaggag
541 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc
601 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca
661 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat
721 gataagctgt caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta
781 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
841 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
901 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
961 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
1021 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
1081 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
1141 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
1201 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
1261 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
1321 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
1381 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
1441 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
1501 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
1561 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
1621 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
1681 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
1741 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
1801 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
1861 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
1921 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
1981 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
2041 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
2101 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
2161 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
2221 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
2281 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
2341 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
2401 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
2461 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
2521 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
2581 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
2641 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
2701 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
2761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
2821 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
2881 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
2941 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
3001 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
3061 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
3121 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
3181 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
3241 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
3301 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
3361 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
3421 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
3481 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
3541 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
3601 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
3661 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
3721 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
3781 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
3841 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
3901 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
3961 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
4021 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
4081 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
4141 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
4201 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
4261 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
4321 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
4381 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
4441 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
4621 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
4681 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
4741 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
4801 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
4861 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
4921 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
4981 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
5041 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
5101 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
5161 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
5221 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
5281 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
5341 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
5401 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
5461 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
5521 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
5581 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
5641 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
5701 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
5761 catg
//
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文献和实验相关实验
中，现在Invitrogen已经克服了这个问题。在载体上多加四个碱基GTGG，就是这四个碱基使它可以与PCR产物定向连接。其作用机理见下图。 结合TOPO技术的有五个系列载体：pET100/D-TOPO®、pET101/D-TOPO®、pET102/D-TOPO®、pET151/D-TOPO®和pET200/D-TOPO®，这五个载体根据所带的标记、是否具有蛋白酶切位点而各有不同。详细信息参见下图。 结合TOPO技术的有五个系列载体：pET100/D
TOPO克隆提供高效的一步法克隆策略，可以定向克隆平端PCR产物到入门载体。平端PCR产物定向克隆的效率>90%，从而简化了筛选。同时不再需要连接酶、PCR后续步骤或者限制性内切酶。目前有两种定向TOPO克隆载体。pENTR/D-TOPO 和pENTR/SD/ D-TOPO（表2和图3）有以下特点： 位于PCR产物插入位点两侧的attL重组位点可以与Gateway目的载体进行有效重组 通用M13位点便于测序 基于pUC的ori位点提供高产量质粒 大肠杆菌中卡那霉素（kanamycin
真DNA聚合酶可能会导致此问题。大肠杆菌 BL21（DE3）中的蛋白质表达重组pET-32α（+）载体可以表达所需的蛋白质，其在被异丙基β-D-硫代半乳糖苷（IPTG）诱导后与大肠杆菌 BL21（DE3）中的109aa Trx•Tag TM融合。首先，将含有100μg/ ml氨苄青霉素的10ml LB培养基与含有重组pET-32α（+）载体的新鲜细菌菌落一起接种37℃过夜; 然后，将500-μl过夜培养物加入到含有100-μg/ ml氨苄青霉素的30-ml新鲜LB培养基中，在37℃接种直至OD 600
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pET100/D-TOPO载体
¥2500
