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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：
pHIS2
- 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：pHIS2酵母单杂交质粒
别称: pHIS2
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 7207 bp ds-DNA circular SYN 23-二月-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7207)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2016年2月23日 from SnapGene Viewer 3.0.3
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7207
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 152..814
/codon_start=1
/gene="S. cerevisiae HIS3"
/product="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/note="HIS3"
/note="yeast auxotrophic marker"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
promoter 2576..2857
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 2858..3532
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
promoter complement(3614..3632)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3653..3669)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3677..3693
/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)."
promoter complement(3701..3731)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3746..3767
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4055..4643)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4814..5608)
/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
GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5609..5713)
/gene="bla"
/note="AmpR promoter"
misc_feature 5746..6247
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
rep_origin complement(6564..7019)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 7164..7180
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 7187..7205
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 gaattcccgg ggagctcacg cgttcgcgaa tcgatccgcg gtctagaaat tcctggcatt
61 atcacataat gaattataca ttatataaag taatgtgatt tcttcgaaga atatactaaa
121 aaatgagcag gcaagataaa cgaaggcaaa gatgacagag cagaaagccc tagtaaagcg
181 tattacaaat gaaaccaaga ttcagattgc gatctcttta aagggtggtc ccctagcgat
241 agagcactcg atcttcccag aaaaagaggc agaagcagta gcagaacagg ccacacaatc
301 gcaagtgatt aacgtccaca caggtatagg gtttctggac catatgatac atgctctggc
361 caagcattcc ggctggtcgc taatcgttga gtgcattggt gacttacaca tagacgacca
421 tcacaccact gaagactgcg ggattgctct cggtcaagct tttaaagagg ccctaggggc
481 cgtgcgtgga gtaaaaaggt ttggatcagg atttgcgcct ttggatgagg cactttccag
541 agcggtggta gatctttcga acaggccgta cgcagttgtc gaacttggtt tgcaaaggga
601 gaaagtagga gatctctctt gcgagatgat cccgcatttt cttgaaagct ttgcagaggc
661 tagcagaatt accctccacg ttgattgtct gcgaggcaag aatgatcatc accgtagtga
721 gagtgcgttc aaggctcttg cggttgccat aagagaagcc acctcgccca atggtaccaa
781 cgatgttccc tccaccaaag gtgttcttat gtagtgacac cgattattta aagctgcagc
841 atacgatata tatacatgtg tatatatgta tacctatgaa tgtcagtaag tatgtatacg
901 aacagtatga tactgaagat gacaaggtaa tgcatcattc tatacgtgtc attctgaacg
961 aggcgcgctt tccttttttc tttttgcttt ttcttttttt ttctcttgaa ctcgagaaaa
1021 aaaatataaa agagatggag gaacgggaaa aagttagttg tggtgatagg tggcaagtgg
1081 tattccgtaa gaacaacaag aaaagcattt catattatgg ctgaactgag cgaacaagtg
1141 caaaatttaa gcatcaacga caacaacgag aatggttatg ttcctcctca cttaagagga
1201 aaaccaagaa gtgccagaaa taacatgagc aactacaata acaacaacgg cggctacaac
1261 ggtggccgtg gcggtggcag cttctttagc aacaaccgtc gtggtggtta cggcaacggt
1321 ggtttcttcg gtggaaacaa cggtggcagc agatctaacg gccgttctgg tggtagatgg
1381 atcgatggca aacatgtccc agctccaaga aacgaaaagg ccgagatcgc catatttggt
1441 gtccccgagg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
1501 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
1561 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
1621 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
1681 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgac cgatgccctt
1741 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
1801 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
1861 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
1921 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
1981 cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct
2041 ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg
2101 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
2161 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat
2221 cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg
2281 cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac
2341 ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga
2401 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
2461 atcgcgtccg ccatctccag cagccgcacg cggcgcatcg gggggggggt ttcaattcaa
2521 ttcatcattt tttttttatt cttttttttg atttcggttt ctttgaaatt tttttaacga
2581 cattactata tatataatat aggaagcatt taatagaaca gcatcgtaat atatgtgtac
2641 tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg
2701 tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc
2761 ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg
2821 agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg
2881 tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc
2941 tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa agagaacaat
3001 tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa atagttcagg
3061 cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg ttttggctct
3121 ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt ggcaagaata
3181 ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat
3241 actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc
3301 aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga
3361 gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga
3421 tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg
3481 tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac
3541 tgagtagtat ttatttaagt attgtttgtg cacttgccct agagtcgacc tgcaggcatg
3601 caagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
3661 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
3721 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
3781 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
3841 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
3901 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
3961 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
4021 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
4081 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
4141 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
4201 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
4261 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
4321 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
4381 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
4441 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
4501 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
4561 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
4621 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
4681 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
4741 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
4801 cttggtctga cagtcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
4861 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
4921 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
4981 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
5041 cgtcatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca
5101 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
5161 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
5221 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
5281 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
5341 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
5401 gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct
5461 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
5521 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
5581 ctgcgtgcaa tccatcttgt tcaatcatac tcttcctttt tcaatattat tgaagcattt
5641 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
5701 taggggttcc gcgcacattt ccccgaaaag tgccacctgc ggacggatcg cttgcctgta
5761 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
5821 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagagtaga agagttacgg
5881 aatgaagaaa aaaaaataaa caaaggttta aaaaatttca acaaaaagcg tactttacat
5941 atatatttat tagacaagaa aagcagatta aatagatata cattcgatta acgataagta
6001 aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat gaaacaattc
6061 ggcattaata cctgagagca ggaagagcaa gataaaaggn agtatttgtt ggcgatcccc
6121 ctagagtctt ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac
6181 tttctatttt taatttatat atttatatta aaaaatttaa attataatta tttttatagc
6241 acgtgatgaa aaggaccgac gtctaagaaa ccattattat catgacatta acctataaaa
6301 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
6361 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
6421 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
6481 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
6541 taaggagaaa ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt
6601 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
6661 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
6721 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
6781 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
6841 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
6901 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
6961 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
7021 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
7081 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
7141 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
7201 atagggc
//
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：pHIS2酵母单杂交质粒
别称: pHIS2
| 启动子: | TRP1 ,T7,T3 |
|---|---|
| 复制子: | pUC |
| 质粒分类: | 酵母系列，酵母单杂交载体 |
| 质粒大小: | 7207bp |
| 原核抗性: | Kan |
| 筛选标记: | TRP1 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 5'测序引物: | M13F:TGTAAAACGACGGCCAGT |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 杂交 |
| 基因种属： | |
| 基因类型： | |
| 原核抗性： | Kan |
| 筛选标记： | TRP1 |
| 荧光蛋白： |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 7207 bp ds-DNA circular SYN 23-二月-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7207)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2016年2月23日 from SnapGene Viewer 3.0.3
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7207
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 152..814
/codon_start=1
/gene="S. cerevisiae HIS3"
/product="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/note="HIS3"
/note="yeast auxotrophic marker"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
promoter 2576..2857
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 2858..3532
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
promoter complement(3614..3632)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3653..3669)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3677..3693
/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)."
promoter complement(3701..3731)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3746..3767
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4055..4643)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4814..5608)
/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
GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5609..5713)
/gene="bla"
/note="AmpR promoter"
misc_feature 5746..6247
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
rep_origin complement(6564..7019)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 7164..7180
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 7187..7205
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 gaattcccgg ggagctcacg cgttcgcgaa tcgatccgcg gtctagaaat tcctggcatt
61 atcacataat gaattataca ttatataaag taatgtgatt tcttcgaaga atatactaaa
121 aaatgagcag gcaagataaa cgaaggcaaa gatgacagag cagaaagccc tagtaaagcg
181 tattacaaat gaaaccaaga ttcagattgc gatctcttta aagggtggtc ccctagcgat
241 agagcactcg atcttcccag aaaaagaggc agaagcagta gcagaacagg ccacacaatc
301 gcaagtgatt aacgtccaca caggtatagg gtttctggac catatgatac atgctctggc
361 caagcattcc ggctggtcgc taatcgttga gtgcattggt gacttacaca tagacgacca
421 tcacaccact gaagactgcg ggattgctct cggtcaagct tttaaagagg ccctaggggc
481 cgtgcgtgga gtaaaaaggt ttggatcagg atttgcgcct ttggatgagg cactttccag
541 agcggtggta gatctttcga acaggccgta cgcagttgtc gaacttggtt tgcaaaggga
601 gaaagtagga gatctctctt gcgagatgat cccgcatttt cttgaaagct ttgcagaggc
661 tagcagaatt accctccacg ttgattgtct gcgaggcaag aatgatcatc accgtagtga
721 gagtgcgttc aaggctcttg cggttgccat aagagaagcc acctcgccca atggtaccaa
781 cgatgttccc tccaccaaag gtgttcttat gtagtgacac cgattattta aagctgcagc
841 atacgatata tatacatgtg tatatatgta tacctatgaa tgtcagtaag tatgtatacg
901 aacagtatga tactgaagat gacaaggtaa tgcatcattc tatacgtgtc attctgaacg
961 aggcgcgctt tccttttttc tttttgcttt ttcttttttt ttctcttgaa ctcgagaaaa
1021 aaaatataaa agagatggag gaacgggaaa aagttagttg tggtgatagg tggcaagtgg
1081 tattccgtaa gaacaacaag aaaagcattt catattatgg ctgaactgag cgaacaagtg
1141 caaaatttaa gcatcaacga caacaacgag aatggttatg ttcctcctca cttaagagga
1201 aaaccaagaa gtgccagaaa taacatgagc aactacaata acaacaacgg cggctacaac
1261 ggtggccgtg gcggtggcag cttctttagc aacaaccgtc gtggtggtta cggcaacggt
1321 ggtttcttcg gtggaaacaa cggtggcagc agatctaacg gccgttctgg tggtagatgg
1381 atcgatggca aacatgtccc agctccaaga aacgaaaagg ccgagatcgc catatttggt
1441 gtccccgagg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
1501 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
1561 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
1621 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
1681 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgac cgatgccctt
1741 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
1801 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
1861 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
1921 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
1981 cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct
2041 ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg
2101 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
2161 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat
2221 cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg
2281 cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac
2341 ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga
2401 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
2461 atcgcgtccg ccatctccag cagccgcacg cggcgcatcg gggggggggt ttcaattcaa
2521 ttcatcattt tttttttatt cttttttttg atttcggttt ctttgaaatt tttttaacga
2581 cattactata tatataatat aggaagcatt taatagaaca gcatcgtaat atatgtgtac
2641 tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg
2701 tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc
2761 ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg
2821 agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg
2881 tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc
2941 tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa agagaacaat
3001 tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa atagttcagg
3061 cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg ttttggctct
3121 ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt ggcaagaata
3181 ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat
3241 actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc
3301 aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga
3361 gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga
3421 tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg
3481 tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac
3541 tgagtagtat ttatttaagt attgtttgtg cacttgccct agagtcgacc tgcaggcatg
3601 caagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
3661 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
3721 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
3781 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
3841 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
3901 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
3961 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
4021 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
4081 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
4141 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
4201 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
4261 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
4321 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
4381 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
4441 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
4501 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
4561 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
4621 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
4681 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
4741 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
4801 cttggtctga cagtcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
4861 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
4921 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
4981 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
5041 cgtcatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca
5101 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
5161 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
5221 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
5281 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
5341 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
5401 gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct
5461 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
5521 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
5581 ctgcgtgcaa tccatcttgt tcaatcatac tcttcctttt tcaatattat tgaagcattt
5641 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
5701 taggggttcc gcgcacattt ccccgaaaag tgccacctgc ggacggatcg cttgcctgta
5761 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
5821 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagagtaga agagttacgg
5881 aatgaagaaa aaaaaataaa caaaggttta aaaaatttca acaaaaagcg tactttacat
5941 atatatttat tagacaagaa aagcagatta aatagatata cattcgatta acgataagta
6001 aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat gaaacaattc
6061 ggcattaata cctgagagca ggaagagcaa gataaaaggn agtatttgtt ggcgatcccc
6121 ctagagtctt ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac
6181 tttctatttt taatttatat atttatatta aaaaatttaa attataatta tttttatagc
6241 acgtgatgaa aaggaccgac gtctaagaaa ccattattat catgacatta acctataaaa
6301 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
6361 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
6421 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
6481 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
6541 taaggagaaa ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt
6601 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
6661 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
6721 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
6781 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
6841 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
6901 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
6961 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
7021 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
7081 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
7141 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
7201 atagggc
//
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
| P2538/pENTR223-SNRPG-G224T人源基因质粒 |
| P2539/pENTR223-SNRPD1-T16G人源基因质粒 |
| P2540/pENTR223-BET1-T337A人源基因质粒 |
| P2541/pENTR223-TMEM14C-T320C人源基因质粒 |
| P2542/pENTR223-COPS7B人源基因质粒 |
| P2543/pENTR223-UBE2D3-C7G人源基因质粒 |
| P2544/pENTR223-TEX11-G1306T-T2761G人源基因质粒 |
| P2545/pENTR223-ATP6V0A1人源基因质粒 |
| P2546/pENTR223-ZNF263人源基因质粒 |
| P2547/pENTR223-ACSL5人源基因质粒 |
| P2548/pENTR223-PRPF3人源基因质粒 |
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pHIS2酵母单杂交质粒
￥100 - 1000
