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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pTet-On
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pTet-On哺乳调控质粒
别称: pTet-On
质粒属性
质粒简介
pTet-On® expresses the reverse tet-responsive transcriptional activator (rtTA) from the strong immediate early promoter of cytomegalovirus (PCMV). tTA is a fusion of amino acids 1–207 of the tet repressor (TetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of herpes simplex virus. pTet-On is similar to pTet-Off except for four amino acid changes that convert the TetR to a rTetR, and consequently, tTA to rtTA. (pTet-Off also contains several silent mutations.) pTet-On was originally described as pUHD17-1neo by Gossen et al. (1995). pTet-On can be distinguished from pTet-Off by digestion with Hind III.
质粒图谱
质粒序列
LOCUS Exported File 7400 bp ds-DNA circular SYN 28-1-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7400)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-1-28 from SnapGene 2.0.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7400
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 89..468
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 469..672
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 774..1781
/codon_start=1
/product="tetracycline-controlled transactivator,
comprising a fusion of the reverse tetracycline repressor
rTetR with the C-terminal activation domain of herpes
simplex virus VP16"
/note="rtTA"
/note="In the Tet-On(R) system, rtTA binds to the
Tet-responsive element?and stimulates transcription only in
the presence of tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLKGESWQDFLRNNAKSFRCALLSHRNGAKVHSDTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALD-MADFEFEQMFTDALGIDEYGG"
polyA_signal 1792..1926
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2646..3234)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3405..4265)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4266..4370)
/gene="bla"
/note="AmpR promoter"
polyA_signal 4483..4617
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(5042..5062)
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/protein_id="
"
/translation="PKKKRKV"
intron 5192..5257
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS complement(5677..6471)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(6832..7161)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6846..6981
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 ctcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt
61 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
721 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt catatgtcta
781 gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg
841 aaggtttaac aacccgtaaa ctcgcccaga agcttggtgt agagcagcct acactgtatt
901 ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc
961 accatactca cttttgccct ttaaaagggg aaagctggca agatttttta cgcaataacg
1021 ctaaaagttt tagatgtgct ttactaagtc atcgcaatgg agcaaaagta cattcagata
1081 cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac
1141 aaggtttttc actagagaac gcgttatatg cactcagcgc tgtggggcat tttactttag
1201 gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta
1261 ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag
1321 agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat
1381 gtgaaagtgg gtccgcgtac agccgcgcgc gtacgaaaaa caattacggg tctaccatcg
1441 agggcctgct cgatctcccg gacgacgacg cccccgaaga ggcggggctg gcggctccgc
1501 gcctgtcctt tctccccgcg ggacacacgc gcagactgtc gacggccccc ccgaccgatg
1561 tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg
1621 cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta
1681 ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga
1741 tgtttaccga tgcccttgga attgacgagt acggtgggta gggggcgcga ggatccagac
1801 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
1861 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
1921 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
1981 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctgcaagc
2041 ctcgtcgtcc tggccggacc acgctatctg tgcaaggtcc ccggccccgg acgcgcgctc
2101 catgagcaga gcgcccgccg ccgaggcgaa gactcgggcg gcgccctgcc cgtcccacca
2161 ggtcaacagg cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg
2221 catgtccccc tggcggacgg gaagtatcca gctcgaccaa gcttggcgag attttcagga
2281 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
2341 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
2401 accgttcagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
2461 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
2521 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
2581 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
2641 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
2701 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
2761 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
2821 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
2881 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
2941 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
3001 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
3061 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
3121 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
3181 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
3241 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
3301 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
3361 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
3421 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
3481 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
3541 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
3601 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
3661 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
3721 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
3781 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
3841 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
3901 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
3961 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
4021 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
4081 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
4141 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
4201 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
4261 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
4321 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
4381 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
4441 aggcgtatca cgaggccctt tcgtcttcac tcgaggtcga gggatccaga catgataaga
4501 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
4561 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
4621 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
4681 agcaagtaaa acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat
4741 acatcaaata ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt
4801 gagcatagtt attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt
4861 tatgattata actgttatgc ctacttataa aggttacaga atatttttcc ataattttct
4921 tgtatagcag tgcagctttt tcctttgtgg tgtaaatagc aaagcaagca agagttctat
4981 tactaaacac agcatgactc aaaaaactta gcaattctga aggaaagtcc ttggggtctt
5041 ctacctttct cttctttttt ggaggagtag aatgttgaga gtcagcagta gcctcatcat
5101 cactagatgg catttcttct gagcaaaaca ggttttcctc attaaaggca ttccaccact
5161 gctcccattc atcagttcca taggttggaa tctaaaatac acaaacaatt agaatcagta
5221 gtttaacaca ttatacactt aaaaatttta tatttacctt agagctttaa atctctgtag
5281 gtagtttgtc caattatgtc acaccacaga agtaaggttc cttcacaaag atccggacca
5341 aagcggccat cgtgcctccc cactcctgca gttcgggggc atggatgcgc ggatagccgc
5401 tgctggtttc ctggatgccg acggatttgc actgccggta gaactccgcg aggtcgtcca
5461 gcctcaggca gcagctgaac caactcgcga ggggatcgag cccggggtgg gcgaagaact
5521 ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga
5581 agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg
5641 tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
5701 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
5761 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
5821 gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac
5881 catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat
5941 gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag
6001 atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt
6061 cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc
6121 agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg
6181 cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc
6241 gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt
6301 cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg
6361 gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct
6421 ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga
6481 tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa
6541 gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg
6601 caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc
6661 actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta
6721 gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt
6781 cctttagcag cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct ttttgcaaaa
6841 gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc
6901 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
6961 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
7021 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt
7081 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
7141 tccacaccct aactgacaca cattccacag ctgcctcgcg cgtttcggtg atgacggtga
7201 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
7261 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
7321 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
7381 attgtactga gagtgcacct
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pTet-On哺乳调控质粒
别称: pTet-On
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal，beta-globin polyA signal |
| 质粒分类: | 哺乳细胞，四环素调控系统载体 |
| 质粒大小: | 7400bp |
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 哺乳细胞 |
| 诱导方式: | 四环素诱导 |
| 5'测序引物: | CMV-F：CGCAAATGGGCGGTAGGCGTG |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： |
质粒简介
pTet-On® expresses the reverse tet-responsive transcriptional activator (rtTA) from the strong immediate early promoter of cytomegalovirus (PCMV). tTA is a fusion of amino acids 1–207 of the tet repressor (TetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of herpes simplex virus. pTet-On is similar to pTet-Off except for four amino acid changes that convert the TetR to a rTetR, and consequently, tTA to rtTA. (pTet-Off also contains several silent mutations.) pTet-On was originally described as pUHD17-1neo by Gossen et al. (1995). pTet-On can be distinguished from pTet-Off by digestion with Hind III.
质粒图谱
质粒序列
LOCUS Exported File 7400 bp ds-DNA circular SYN 28-1-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7400)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-1-28 from SnapGene 2.0.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7400
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 89..468
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 469..672
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 774..1781
/codon_start=1
/product="tetracycline-controlled transactivator,
comprising a fusion of the reverse tetracycline repressor
rTetR with the C-terminal activation domain of herpes
simplex virus VP16"
/note="rtTA"
/note="In the Tet-On(R) system, rtTA binds to the
Tet-responsive element?and stimulates transcription only in
the presence of tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLKGESWQDFLRNNAKSFRCALLSHRNGAKVHSDTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALD-MADFEFEQMFTDALGIDEYGG"
polyA_signal 1792..1926
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2646..3234)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3405..4265)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4266..4370)
/gene="bla"
/note="AmpR promoter"
polyA_signal 4483..4617
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(5042..5062)
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/protein_id="
"
/translation="PKKKRKV"
intron 5192..5257
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS complement(5677..6471)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(6832..7161)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6846..6981
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 ctcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt
61 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
721 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt catatgtcta
781 gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg
841 aaggtttaac aacccgtaaa ctcgcccaga agcttggtgt agagcagcct acactgtatt
901 ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc
961 accatactca cttttgccct ttaaaagggg aaagctggca agatttttta cgcaataacg
1021 ctaaaagttt tagatgtgct ttactaagtc atcgcaatgg agcaaaagta cattcagata
1081 cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac
1141 aaggtttttc actagagaac gcgttatatg cactcagcgc tgtggggcat tttactttag
1201 gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta
1261 ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag
1321 agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat
1381 gtgaaagtgg gtccgcgtac agccgcgcgc gtacgaaaaa caattacggg tctaccatcg
1441 agggcctgct cgatctcccg gacgacgacg cccccgaaga ggcggggctg gcggctccgc
1501 gcctgtcctt tctccccgcg ggacacacgc gcagactgtc gacggccccc ccgaccgatg
1561 tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg
1621 cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta
1681 ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga
1741 tgtttaccga tgcccttgga attgacgagt acggtgggta gggggcgcga ggatccagac
1801 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
1861 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
1921 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
1981 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctgcaagc
2041 ctcgtcgtcc tggccggacc acgctatctg tgcaaggtcc ccggccccgg acgcgcgctc
2101 catgagcaga gcgcccgccg ccgaggcgaa gactcgggcg gcgccctgcc cgtcccacca
2161 ggtcaacagg cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg
2221 catgtccccc tggcggacgg gaagtatcca gctcgaccaa gcttggcgag attttcagga
2281 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
2341 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
2401 accgttcagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
2461 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
2521 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
2581 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
2641 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
2701 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
2761 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
2821 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
2881 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
2941 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
3001 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
3061 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
3121 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
3181 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
3241 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
3301 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
3361 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
3421 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
3481 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
3541 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
3601 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
3661 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
3721 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
3781 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
3841 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
3901 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
3961 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
4021 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
4081 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
4141 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
4201 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
4261 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
4321 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
4381 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
4441 aggcgtatca cgaggccctt tcgtcttcac tcgaggtcga gggatccaga catgataaga
4501 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
4561 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
4621 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
4681 agcaagtaaa acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat
4741 acatcaaata ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt
4801 gagcatagtt attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt
4861 tatgattata actgttatgc ctacttataa aggttacaga atatttttcc ataattttct
4921 tgtatagcag tgcagctttt tcctttgtgg tgtaaatagc aaagcaagca agagttctat
4981 tactaaacac agcatgactc aaaaaactta gcaattctga aggaaagtcc ttggggtctt
5041 ctacctttct cttctttttt ggaggagtag aatgttgaga gtcagcagta gcctcatcat
5101 cactagatgg catttcttct gagcaaaaca ggttttcctc attaaaggca ttccaccact
5161 gctcccattc atcagttcca taggttggaa tctaaaatac acaaacaatt agaatcagta
5221 gtttaacaca ttatacactt aaaaatttta tatttacctt agagctttaa atctctgtag
5281 gtagtttgtc caattatgtc acaccacaga agtaaggttc cttcacaaag atccggacca
5341 aagcggccat cgtgcctccc cactcctgca gttcgggggc atggatgcgc ggatagccgc
5401 tgctggtttc ctggatgccg acggatttgc actgccggta gaactccgcg aggtcgtcca
5461 gcctcaggca gcagctgaac caactcgcga ggggatcgag cccggggtgg gcgaagaact
5521 ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga
5581 agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg
5641 tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
5701 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
5761 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
5821 gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac
5881 catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat
5941 gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag
6001 atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt
6061 cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc
6121 agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg
6181 cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc
6241 gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt
6301 cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg
6361 gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct
6421 ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga
6481 tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa
6541 gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg
6601 caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc
6661 actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta
6721 gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt
6781 cctttagcag cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct ttttgcaaaa
6841 gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc
6901 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
6961 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
7021 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt
7081 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
7141 tccacaccct aactgacaca cattccacag ctgcctcgcg cgtttcggtg atgacggtga
7201 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
7261 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
7321 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
7381 attgtactga gagtgcacct
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P2755/pENTR223-SPRY2人源基因质粒 |
| P2756/pENTR223-CYB5R1人源基因质粒 |
| P2757/pENTR223-RGR人源基因质粒 |
| P2758/pENTR223-ANKHD1人源基因质粒 |
| P2765/pENTR223-AGPAT2-G598T人源基因质粒 |
| P2767/pENTR223-RNF13人源基因质粒 |
| P2768/pENTR223-EXOSC3（1同义突变）人源基因质粒 |
| P2769/pENTR223-KIAA1257人源基因质粒 |
| P2770/pENTR223-WDR61人源基因质粒 |
| P2771/pENTR223-MRPL37人源基因质粒 |
| P2772/pENTR223-SPATA6L（有突变）人源基因质粒 |
| P2773/pENTR223-DDI1（有突变）人源基因质粒 |
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pTet-On哺乳调控质粒
￥100 - 1000
