| 出品公司: | Invitrogen |
|---|---|
| 载体名称: | pET101 ； pET101/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: | 5753 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: | V5 tag、6xHis 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: | pET101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: | ZY100-02 |
| 组成型/诱导型: | 诱导型 (IPTG) |
| 病毒/非病毒: | 非病毒 |
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- ZY100-02
- 2025年07月16日
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- 详细信息
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- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pET101/D-TOPO
- 库存：
20
- 供应商：
泽叶生物
pET101载体基本信息
pET101载体质粒图谱和多克隆位点信息
pET101载体简介
pET101载体序列
LOCUS pET101-D-TOPO 5753 bp ds-DNA circular SYN 15-AUG-2016 DEFINITION synthetic circular DNA KEYWORDS pET101-D-TOPO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5753) AUTHORS Biofeng Lab. FEATURES Location/Qualifiers source 1..5753 /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)." misc_feature 282..296 /note="RBS" misc_feature 282..288 /note="RBS" misc_feature 293..296 /note="RBS" misc_feature 297..310 /note="TOPO site" CDS 333..374 /codon_start=1 /product="epitope tag from simian virus 5" /note="V5 tag" /translation="GKPIPNPLLGLDST" CDS 384..401 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" terminator 416..544 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(694..722) /note="tet promoter" /note="E. coli promoter for tetracycline efflux protein gene" promoter 845..943 /gene="bla" /note="bla promoter" CDS 944..1804 /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 1949..2622 /direction=RIGHT /note="pBR322" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 2749..2888 /note="bom" /note="basis of mobility region from pBR322" CDS complement(2990..3181) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4493..5584) /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(5576..5653) /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 agaaggaatt caggagccct 301 tcaccaaggg cgagctcaat tcgaagcttg aaggtaagcc tatccctaac cctctcctcg 361 gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttga tccggctgct 421 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa 481 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 541 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca 601 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac 661 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc 721 aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt 781 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 841 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 901 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 961 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 1021 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 1081 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 1141 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 1201 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 1261 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 1321 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 1381 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 1441 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac 1501 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 1561 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 1621 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 1681 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 1741 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 1801 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 1861 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 1921 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 1981 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 2041 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 2101 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 2161 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 2221 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 2281 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 2341 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 2401 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 2461 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 2521 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 2581 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 2641 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 2701 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta 2761 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc 2821 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca 2881 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2941 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 3001 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa 3061 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg 3121 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca 3181 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag 3241 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg 3301 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat 3361 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga 3421 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa 3481 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt 3541 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc 3601 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa 3661 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct 3721 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg 3781 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct 3841 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca 3901 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc 3961 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc 4021 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc 4081 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta 4141 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt 4201 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga 4261 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc 4321 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat 4381 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg 4441 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc 4501 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4561 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca 4621 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg 4681 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc 4741 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact 4801 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg 4861 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt 4921 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag 4981 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt 5041 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa 5101 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg 5161 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga 5221 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta 5281 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa 5341 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt 5401 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt 5461 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg 5521 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca 5581 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc 5641 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag 5701 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atg
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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
pECFP-C pEGFP-C-5’ pECFP-C-3' pECFP-N1 pEGFP-N-5’ pEGFP-N-3’ pEF/myc/ER pEF-F pCDNA3.1R pEGFP-C(KAN+) pEGFP-C-5’ pEGFP-C-3' pEGFP-N(KAN+) pEGFP-N-5’ pEGFP-N-3’ pENTR/D-TOPO(KAN+) M13F M13R pET-*(His)(KAN+) T7 T7 Ter pET22(a,b,c)(AMP+) T7 T7 Ter
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pET101/D-TOPO载体
¥2500
