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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：
pRS423
- 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：pRS423酿酒酵母质粒
别称: pRS423
质粒属性
质粒简介
A YEtype(episomal) E. coli/S. cerevisiae shuttle vector permitting RNA synthesis in vitro, visual detection of recombinants, production of single stranded DNA and containing primer sites useful for sequencing Designation: pRS423
质粒图谱
质粒序列
ACCESSION .
VERSION .
KEYWORDS pRS423
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5797)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5797
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 68..1410
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind 400..447
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 1437..1541
/gene="bla"
/note="AmpR promoter"
CDS 1542..2402
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2573..3161
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 3485..3515
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3523..3539
/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)."
primer_bind 3547..3563
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 3559..4137
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
promoter 3584..3602
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 3615..3722
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 3639..3655
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3689..3705)
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3731..3749)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3759..3775)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3920..4375
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(4635..5294)
/codon_start=1
/gene="S. cerevisiae HIS3"
/product="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/note="HIS3"
/note="yeast auxotrophic marker"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
promoter complement(5295..5481)
/gene="S. cerevisiae HIS3"
/note="HIS3 promoter"
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga
121 ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc
181 atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg
241 catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat
301 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg
361 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc
421 ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag
481 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
541 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
601 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
661 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
721 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
781 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
841 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
901 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
961 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
1021 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
1081 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
1141 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
1201 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
1261 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
1321 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
1381 ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg
1441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
1501 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
1561 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
1621 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
1681 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
1741 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
1801 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
1861 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
1921 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
1981 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
2041 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
2101 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
2161 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
2221 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
2281 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
2341 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
2401 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
2461 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
2521 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
2581 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
2641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
2701 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
2761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
2821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
2881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
2941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
3001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
3061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
3121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
3181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
3241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
3301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
3421 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat taggcacccc
3481 aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc ggataacaat
3541 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
3601 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatcccccg
3661 ggctgcagga attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta
3721 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
3781 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
3841 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
3901 ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
3961 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
4021 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
4081 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
4141 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
4201 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
4261 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
4321 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcctga
4381 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat ccgtcgagtt
4441 caagagaaaa aaaaagaaaa agcaaaaaga aaaaaggaaa gcgcgcctcg ttcagaatga
4501 cacgtataga atgatgcatt accttgtcat cttcagtatc atactgttcg tatacatact
4561 tactgacatt cataggtata catatataca catgtatata tatcgtatgc tgcagcttta
4621 aataatcggt gtcactacat aagaacacct ttggtggagg gaacatcgtt ggtaccattg
4681 ggcgaggtgg cttctcttat ggcaaccgca agagccttga acgcactctc actacggtga
4741 tgatcattct tgcctcgcag acaatcaacg tggagggtaa ttctgctagc ctctgcaaag
4801 ctttcaagaa aatgcgggat catctcgcaa gagagatctc ctactttctc cctttgcaaa
4861 ccaagttcga caactgcgta cggcctgttc gaaagatcta ccaccgctct ggaaagtgcc
4921 tcatccaaag gcgcaaatcc tgatccaaac ctttttactc cacgcgccag tagggcctct
4981 ttaaaagctt gaccgagagc aatcccgcag tcttcagtgg tgtgatggtc gtctatgtgt
5041 aagtcaccaa tgcactcaac gattagcgac cagccggaat gcttggccag agcatgtatc
5101 atatggtcca gaaaccctat acctgtgtgg acgttaatca cttgcgattg tgtggcctgt
5161 tctgctactg cttctgcctc tttttctggg aagatcgagt gctctatcgc taggggacca
5221 ccctttaaag agatcgcaat ctgaatcttg gtttcatttg taatacgctt tactagggct
5281 ttctgctctg tcatctttgc cttcgtttat cttgcctgct cattttttag tatattcttc
5341 gaagaaatca cattacttta tataatgtat aattcattat gtgataatgc caatcgctaa
5401 gaaaaaaaaa gagtcatccg ctaggggaaa aaaaaaaatg aaaatcatta ccgaggcata
5461 aaaaaatata gagtgtacta gaggaggcca agagtaatag aaaaagaaaa ttgcgggaaa
5521 ggactgtgtt atgacttccc tgactaatgc cgtgttcaaa cgatacctgg cagtgactcc
5581 tagcgctcac caagctctta aaacgggaat ttatggtgca ctctcagtac aatctgctct
5641 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
5701 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
5761 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
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：pRS423酿酒酵母质粒
别称: pRS423
| 复制子: | 2μFI |
|---|---|
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 5797bp |
| 原核抗性: | Amp |
| 真核抗性: | HIS3 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 5'测序引物: | T7:TAATACGACTCACTATAGGG |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 质粒宿主： | 酵母菌 |
|---|---|
| 质粒用途： | 蛋白表达 |
| 片段类型： | |
| 片段物种： | |
| 原核抗性： | Amp |
| 真核抗性： | HIS3 |
| 荧光标记： |
质粒简介
A YEtype(episomal) E. coli/S. cerevisiae shuttle vector permitting RNA synthesis in vitro, visual detection of recombinants, production of single stranded DNA and containing primer sites useful for sequencing Designation: pRS423
质粒图谱
质粒序列
LOCUS Exported 5797 bp ds-DNA circular SYN 25-MAY-2016
DEFINITION synthetic circular DNAACCESSION .
VERSION .
KEYWORDS pRS423
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5797)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5797
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 68..1410
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind 400..447
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 1437..1541
/gene="bla"
/note="AmpR promoter"
CDS 1542..2402
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2573..3161
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 3485..3515
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3523..3539
/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)."
primer_bind 3547..3563
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 3559..4137
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
promoter 3584..3602
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 3615..3722
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 3639..3655
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3689..3705)
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3731..3749)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3759..3775)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3920..4375
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(4635..5294)
/codon_start=1
/gene="S. cerevisiae HIS3"
/product="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/note="HIS3"
/note="yeast auxotrophic marker"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
promoter complement(5295..5481)
/gene="S. cerevisiae HIS3"
/note="HIS3 promoter"
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga
121 ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc
181 atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg
241 catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat
301 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg
361 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc
421 ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag
481 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
541 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
601 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
661 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
721 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
781 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
841 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
901 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
961 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
1021 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
1081 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
1141 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
1201 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
1261 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
1321 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
1381 ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg
1441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
1501 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
1561 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
1621 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
1681 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
1741 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
1801 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
1861 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
1921 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
1981 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
2041 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
2101 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
2161 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
2221 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
2281 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
2341 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
2401 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
2461 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
2521 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
2581 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
2641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
2701 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
2761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
2821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
2881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
2941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
3001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
3061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
3121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
3181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
3241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
3301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
3421 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat taggcacccc
3481 aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc ggataacaat
3541 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
3601 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatcccccg
3661 ggctgcagga attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta
3721 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
3781 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
3841 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
3901 ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
3961 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
4021 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
4081 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
4141 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
4201 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
4261 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
4321 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcctga
4381 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat ccgtcgagtt
4441 caagagaaaa aaaaagaaaa agcaaaaaga aaaaaggaaa gcgcgcctcg ttcagaatga
4501 cacgtataga atgatgcatt accttgtcat cttcagtatc atactgttcg tatacatact
4561 tactgacatt cataggtata catatataca catgtatata tatcgtatgc tgcagcttta
4621 aataatcggt gtcactacat aagaacacct ttggtggagg gaacatcgtt ggtaccattg
4681 ggcgaggtgg cttctcttat ggcaaccgca agagccttga acgcactctc actacggtga
4741 tgatcattct tgcctcgcag acaatcaacg tggagggtaa ttctgctagc ctctgcaaag
4801 ctttcaagaa aatgcgggat catctcgcaa gagagatctc ctactttctc cctttgcaaa
4861 ccaagttcga caactgcgta cggcctgttc gaaagatcta ccaccgctct ggaaagtgcc
4921 tcatccaaag gcgcaaatcc tgatccaaac ctttttactc cacgcgccag tagggcctct
4981 ttaaaagctt gaccgagagc aatcccgcag tcttcagtgg tgtgatggtc gtctatgtgt
5041 aagtcaccaa tgcactcaac gattagcgac cagccggaat gcttggccag agcatgtatc
5101 atatggtcca gaaaccctat acctgtgtgg acgttaatca cttgcgattg tgtggcctgt
5161 tctgctactg cttctgcctc tttttctggg aagatcgagt gctctatcgc taggggacca
5221 ccctttaaag agatcgcaat ctgaatcttg gtttcatttg taatacgctt tactagggct
5281 ttctgctctg tcatctttgc cttcgtttat cttgcctgct cattttttag tatattcttc
5341 gaagaaatca cattacttta tataatgtat aattcattat gtgataatgc caatcgctaa
5401 gaaaaaaaaa gagtcatccg ctaggggaaa aaaaaaaatg aaaatcatta ccgaggcata
5461 aaaaaatata gagtgtacta gaggaggcca agagtaatag aaaaagaaaa ttgcgggaaa
5521 ggactgtgtt atgacttccc tgactaatgc cgtgttcaaa cgatacctgg cagtgactcc
5581 tagcgctcac caagctctta aaacgggaat ttatggtgca ctctcagtac aatctgctct
5641 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
5701 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
5761 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
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
| P2040/pENTR223-MOBKL2A人源基因质粒 |
| P2041/pENTR223-SAMD3人源基因质粒 |
| P2042/pENTR223-MAGEH1人源基因质粒 |
| P2043/pENTR223-CLTA人源基因质粒 |
| P2057/pENTR223-C22orf23人源基因质粒 |
| P2058/pENTR223-TMED3人源基因质粒 |
| P2059/pENTR223-HEATR2-C13G人源基因质粒 |
| P2060/pENTR223-TWSG1人源基因质粒 |
| P2061/pENTR223-TMEM86B人源基因质粒 |
| P2062/pENTR223-NUDCD3人源基因质粒 |
| P2063/pENTR223-TRAPPC4人源基因质粒 |
| P2064/pENTR223-GJB2人源基因质粒 |
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pRS423酿酒酵母质粒
￥100 - 1000
