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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：
pIEFBPR
- 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：pIEFBPR枯草分泌质粒
别称: pIEFBPR
质粒属性
质粒简介
pIEFBPR质粒是一个枯草整合型分泌载体，
质粒图谱
质粒序列
LOCUS Exported 8965 bp ds-DNA circular SYN 15-SEP-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8965)
TITLE Direct Submission
JOURNAL Exported Sep 15, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8965
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 4..459
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 626..644
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 692..1697
/label=bpr-front
/note="upstream of Bacillus subtilis bpr gene"
misc_feature 1728..1852
/label=DR sequence
CDS complement(1900..2982)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
CDS complement(3232..3567)
/codon_start=1
/label=mazF
/note="Escherichia coli toxin MazF"
/translation="MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGM
CLCVPCTTQSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKA
KINVLIG"
CDS 4179..4931
/codon_start=1
/product="spectinomycin adenyltransferase; selectable
either in Escherichia coli or Bacillus subtilis"
/label=spc
/translation="LNTYEQINKVKKILRKHLKNNLIGTYMFGSGVESGLKPNSDLDFL
VVVSEPLTDQSKEILIQKIRPISKKIGDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYG
EWLQELYEQGYIPQKELNSDLTIMLYQAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMD
SSEELIDNYQDDETNSILTLCRMILTMDTGKIIPKDIAGNAVAESSPLEHRERILLAVR
SYLGENIEWTNENVNLTINYLNNRLKKL"
misc_feature 4933..5057
/label=DR sequence
misc_feature 5197..6235
/label=bpr-back
/note="downstream of Bacillus subtilis bpr gene; homologous
arm for recombination"
misc_feature 6450..6758
/label=mazE
CDS complement(7218..7806)
/codon_start=1
/label=ColE1 ori
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
/translation="LRSFFSARNLLLANKKTTATSGGLFAGSRATNSFSEGNWLQQSAD
TKYCPSSVAVVRPPLQELCSTAYIPRSANPVTSGCCQWR*VVSYRVGLKTIVTG*GAAV
GLNGGFVHTAQLGANDLHRTEIPTA*AMRKRHASRREKGGQVSGKRQGRNRRAHEGASR
GKRLVSL*SCRVSPPLT*ASIFVMLVRGAEPME"
CDS complement(7977..8837)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8838..8942)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
661 gccccccctc gagttaacct gcaggcgcgc ctttgatggg tatcggtatt gctactgggg
721 aaaatcgcgc ggcagaggca gcaaaaaaag caatttccag cccgcttctt gaagcggcca
781 ttgacggtgc gcaaggcgtc ctcatgaaca tcactggagg aacaaacctc agcctatatg
841 aggttcagga agcagcagac attgtcgctt cggcgtctga tcaagacgta aacatgattt
901 tcggttctgt tattaatgaa aatctaaaag atgagattgt ggtgacagtg attgcaaccg
961 gctttatcga acaagagaag gacgtgacga agcctcagcg cccaagctta aatcaaagca
1021 tcaaaacaca caatcaaagt gttccgaagc gtgacgcaaa acgtgaggaa cctcagcagc
1081 agaacacagt aagccgtcat acttcacagc cggctgatga tacgcttgac atcccgacat
1141 tcttaagaaa ccgtaataaa cgcggctaat gtaaaggaca aaatcgtttt cgattttgtc
1201 ttttttgttt ttctcttcac acttccttct tataaagtct ttttccctat tgctttcctt
1261 cgcttagtaa caaaacagat aattagaccc atttattttt gtgacatttt tatcattttc
1321 atatatatgg aaattgaatg acatgaaacg acaatatctg taattcagat tgtctacagt
1381 taatatacag cgatgttctg acaaaccatt cattattaaa aggagggacg acactttttt
1441 taaaaagcat gttgaaaaag ggggatgaaa atgaggaaaa aaacgaaaaa cagactcatc
1501 agctctgttt taagtacagt tgtcatcagt tcactgctgt ttccgggagc agccggggca
1561 agcagtaaag tcacctcacc ttctgttaaa aaggagcttc aatctgcgga atccattcaa
1621 aacaagattt cgagttcatt aaagaaaagc tttaaaaaga aagaaaaaac gacttttctg
1681 attaaattta aagatctatc gatgctagcc atggtcgacg cggccgcaaa attgaaaaaa
1741 tggtggaaac acttttttca atttttttgt tttattattt aatatttggg aaatattcat
1801 tctaattggt aatcagattt tagaaaacaa taaacccttg cataggggga tctccggagt
1861 ttaaaccgcg gatcctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
1921 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
1981 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
2041 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
2101 gcgaaaatcc tgtttgatgg tggttgacgg cgggatataa catgagctgt cttcggtatc
2161 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
2221 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
2281 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
2341 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
2401 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
2461 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
2521 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
2581 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
2641 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
2701 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
2761 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
2821 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
2881 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
2941 accggcatac tctgcgacat cgtataacgt tactggtttc atcaaaatcg tctccctccg
3001 tttgaatatt tgattgatcg taaccagatg aagcactctt tccactatcc ctacagtgtt
3061 atggcttgaa caatcacgaa acaataattg gtacgtacga tctttcagcc gactcaaaca
3121 tcaaatctta caaatgtagt ctttgaaagt attacatatg taagatttaa atgcaaccgt
3181 tttttcggaa ggaaatgatg acctcgtttc caccggaatt agcttgcatg cctacccaat
3241 cagtacgtta attttggctt taatgagttg taattcctct ggggcaactg ttcctttctt
3301 cgttgctcct cttgcccgcc aggcgatact ttttacctga tcagctaacg ctacgccatc
3361 acgttcctga ccggataaaa caacttcgaa cggatatcct tttgattgcg ttgtacaagg
3421 aacacacaga cacatacctg ttttgttgtt gtacatgaaa ggactcagga caacagctgg
3481 acgatgtcca gcttgctcgc taccttttgt cgggtcaaaa tcaacccaaa tcagatcgcc
3541 catatcgggt acgtatcggc ttaccatacc tgcttcctcc ttaagcttaa ttgttatccg
3601 ctcacaattc cacacattat gccacacctt gtagataaag tcaacaactt tttgcaaaat
3661 ttttcaggaa ttttagcaga ggttgttctg gatgtagaac aaaacatctt tccgctcttg
3721 tgctgttagg atatctttct tggaagctag gtaggcaagg gctacctcaa ataaatcttc
3781 ttcagggtgc gctattttta aggtgcctag tgaggtcttg accacttcac ccataatttc
3841 agtgccgaat agtctggact gggctgtgta gaattcctgc agccctggcg aatggcgatt
3901 ttcgttcgtg aatacatgtt ataataacta taactaataa cgtaacgtga ctggcaagag
3961 atatttttaa aacaatgaat aggtttacac ttactttagt tttatggaaa tgaaagatca
4021 tatcatatat aatctagaat aaaattaact aaaataatta ttatctagat aaaaaattta
4081 gaagccaatg aaatctataa ataaactaaa ttaagtttat ttaattaaca actatggata
4141 taaaataggt actaatcaaa atagtgagga ggatatattt gaatacatac gaacaaatta
4201 ataaagtgaa aaaaatactt cggaaacatt taaaaaataa ccttattggt acttacatgt
4261 ttggatcagg agttgagagt ggactaaaac caaatagtga tcttgacttt ttagtcgtcg
4321 tatctgaacc attgacagat caaagtaaag aaatacttat acaaaaaatt agacctattt
4381 caaaaaaaat aggagataaa agcaacttac gatatattga attaacaatt attattcagc
4441 aagaaatggt accgtggaat catcctccca aacaagaatt tatttatgga gaatggttac
4501 aagagcttta tgaacaagga tacattcctc agaaggaatt aaattcagat ttaaccataa
4561 tgctttacca agcaaaacga aaaaataaaa gaatatacgg aaattatgac ttagaggaat
4621 tactacctga tattccattt tctgatgtga gaagagccat tatggattcg tcagaggaat
4681 taatagataa ttatcaggat gatgaaacca actctatatt aactttatgc cgtatgattt
4741 taactatgga cacgggtaaa atcataccaa aagatattgc gggaaatgca gtggctgaat
4801 cttctccatt agaacatagg gagagaattt tgttagcagt tcgtagttat cttggagaga
4861 atattgaatg gactaatgaa aatgtaaatt taactataaa ctatttaaat aacagattaa
4921 aaaaattata aaaaaattga aaaaatggtg gaaacacttt tttcaatttt tttgttttat
4981 tatttaatat ttgggaaata ttcattctaa ttggtaatca gattttagaa aacaataaac
5041 ccttgcatag ggggatctcg acatggatga gcgatgatga tatccgttta ggctgggcgg
5101 tgatagcttc tcgttcaggc agtacgcctc ttttcttttc cagacctgag ggaggcggaa
5161 atggtgtgag gttcccgggc cggccaattg atgcatcagt tcaaaaggga ttatggtcga
5221 taaagatttt tatctcgtat atatccagtc gaaacctgat ccgtattcac ctggattggc
5281 aatggatgaa accggccaga attccggccg caactggcag tatatagatg gaaagtggca
5341 gccaggtgac aaagcggatg gcaactatat gattcgcgca ttagttgatt atgaagctgc
5401 tgtacctgag attacttcac cgacagacaa atcatacaca aataaggata gcgtcactgt
5461 aaaaggaaac gcttctcctg gcacaacggt acacatttat aatggagaga aagaagcagg
5521 agaaacgaaa gctgctgcgg atggcacgtt ccatgcaggc atcatactca acaagggtga
5581 aaatgagctg acggcaactg catcaactga caacggaaca acagatgcct ccagcccaat
5641 cacggtcacg cttgatcaag aaaagcctga attaacactg gacaatccaa aggatggcgg
5701 gaaaacaaat aaagaaacgc tgactgtcaa aggggctgta tccgatgaca atctgaaaga
5761 cgtcaaggtg aatggcaaaa aagcaacagt agctgatggt tcatactcag cccgtattct
5821 tttggaaaat ggaagaaatg aaatcaaggt aattgctaca gacttggcag gcaacaaaac
5881 gacgaaaaag acagtcattg atgtgaactt tgacaagcct gtcatttccg gcttaattcc
5941 gggagaggat aaaaacttaa aagccggtga atctgtgaaa atcgctttct caagcgctga
6001 ggatttagat gcaacgttta ccattcgtat gccgctgacc aatgcaagag cgagtgtgca
6061 aaatgccacc gaactcccgt taagagaaat ctctccgggg agatatgaag gctattggac
6121 tgccacttct tctattaaag caaaaggagc aaaagtagaa gtgatcgtcc gagatgatta
6181 tggaaatgaa acaagaaaaa ctgcgaatgg aaaacttaat atgaacacag aaaataccgg
6241 tcctagggcg atcgcactag tcgataacca gaagcaataa aaaatcaaat cggatttcac
6301 tatataatct cactttatct aagatgaatc cgatggaagc atcctgtttt ctctcaattt
6361 ttttatctaa aacccagcgt tcgatgcttc tttgagcgaa cgatcaaaaa taagtgcctt
6421 cccatcaaaa aaatattctc aacataaaaa actttgtgta atacttgtaa cgctacatgg
6481 agattaactc aatctagagg gtattaataa tgatccacag tagcgtaaag cgttggggaa
6541 attcaccggc ggtgcggatt ccggctacgt taatgcaggc gctcaatctg aatattgatg
6601 atgaagtgaa gattgacctg gtggatggca aattaattat tgagccagtg cgtaaagagc
6661 ccgtatttac gcttgctgaa ctggtcaacg acatcacgcc ggaaaacctc cacgagaata
6721 tcgactgggg agagccgaaa gataaggaag tctggtaaga gctccagctt ttgttccctt
6781 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
6841 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
6901 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
6961 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
7021 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
7081 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
7141 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
7201 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
7261 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
7321 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
7381 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
7441 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
7501 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
7561 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
7621 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
7681 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
7741 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
7801 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
7861 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
7921 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
7981 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
8041 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
8101 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
8161 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
8221 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
8281 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
8341 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
8401 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
8461 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
8521 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
8581 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
8641 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
8701 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
8761 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
8821 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
8881 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
8941 cgcacatttc cccgaaaagt gccac
//
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：pIEFBPR枯草分泌质粒
别称: pIEFBPR
| 复制子: | ColE1 Ori |
|---|---|
| 质粒分类: | 枯草杆菌质粒；枯草整合质粒；枯草分泌质粒 |
| 质粒大小: | 8965bp |
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 枯草杆菌 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | |
| 荧光蛋白： |
质粒简介
pIEFBPR质粒是一个枯草整合型分泌载体，
质粒图谱
质粒序列
LOCUS Exported 8965 bp ds-DNA circular SYN 15-SEP-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8965)
TITLE Direct Submission
JOURNAL Exported Sep 15, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8965
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 4..459
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 626..644
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 692..1697
/label=bpr-front
/note="upstream of Bacillus subtilis bpr gene"
misc_feature 1728..1852
/label=DR sequence
CDS complement(1900..2982)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
CDS complement(3232..3567)
/codon_start=1
/label=mazF
/note="Escherichia coli toxin MazF"
/translation="MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGM
CLCVPCTTQSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKA
KINVLIG"
CDS 4179..4931
/codon_start=1
/product="spectinomycin adenyltransferase; selectable
either in Escherichia coli or Bacillus subtilis"
/label=spc
/translation="LNTYEQINKVKKILRKHLKNNLIGTYMFGSGVESGLKPNSDLDFL
VVVSEPLTDQSKEILIQKIRPISKKIGDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYG
EWLQELYEQGYIPQKELNSDLTIMLYQAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMD
SSEELIDNYQDDETNSILTLCRMILTMDTGKIIPKDIAGNAVAESSPLEHRERILLAVR
SYLGENIEWTNENVNLTINYLNNRLKKL"
misc_feature 4933..5057
/label=DR sequence
misc_feature 5197..6235
/label=bpr-back
/note="downstream of Bacillus subtilis bpr gene; homologous
arm for recombination"
misc_feature 6450..6758
/label=mazE
CDS complement(7218..7806)
/codon_start=1
/label=ColE1 ori
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
/translation="LRSFFSARNLLLANKKTTATSGGLFAGSRATNSFSEGNWLQQSAD
TKYCPSSVAVVRPPLQELCSTAYIPRSANPVTSGCCQWR*VVSYRVGLKTIVTG*GAAV
GLNGGFVHTAQLGANDLHRTEIPTA*AMRKRHASRREKGGQVSGKRQGRNRRAHEGASR
GKRLVSL*SCRVSPPLT*ASIFVMLVRGAEPME"
CDS complement(7977..8837)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8838..8942)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
661 gccccccctc gagttaacct gcaggcgcgc ctttgatggg tatcggtatt gctactgggg
721 aaaatcgcgc ggcagaggca gcaaaaaaag caatttccag cccgcttctt gaagcggcca
781 ttgacggtgc gcaaggcgtc ctcatgaaca tcactggagg aacaaacctc agcctatatg
841 aggttcagga agcagcagac attgtcgctt cggcgtctga tcaagacgta aacatgattt
901 tcggttctgt tattaatgaa aatctaaaag atgagattgt ggtgacagtg attgcaaccg
961 gctttatcga acaagagaag gacgtgacga agcctcagcg cccaagctta aatcaaagca
1021 tcaaaacaca caatcaaagt gttccgaagc gtgacgcaaa acgtgaggaa cctcagcagc
1081 agaacacagt aagccgtcat acttcacagc cggctgatga tacgcttgac atcccgacat
1141 tcttaagaaa ccgtaataaa cgcggctaat gtaaaggaca aaatcgtttt cgattttgtc
1201 ttttttgttt ttctcttcac acttccttct tataaagtct ttttccctat tgctttcctt
1261 cgcttagtaa caaaacagat aattagaccc atttattttt gtgacatttt tatcattttc
1321 atatatatgg aaattgaatg acatgaaacg acaatatctg taattcagat tgtctacagt
1381 taatatacag cgatgttctg acaaaccatt cattattaaa aggagggacg acactttttt
1441 taaaaagcat gttgaaaaag ggggatgaaa atgaggaaaa aaacgaaaaa cagactcatc
1501 agctctgttt taagtacagt tgtcatcagt tcactgctgt ttccgggagc agccggggca
1561 agcagtaaag tcacctcacc ttctgttaaa aaggagcttc aatctgcgga atccattcaa
1621 aacaagattt cgagttcatt aaagaaaagc tttaaaaaga aagaaaaaac gacttttctg
1681 attaaattta aagatctatc gatgctagcc atggtcgacg cggccgcaaa attgaaaaaa
1741 tggtggaaac acttttttca atttttttgt tttattattt aatatttggg aaatattcat
1801 tctaattggt aatcagattt tagaaaacaa taaacccttg cataggggga tctccggagt
1861 ttaaaccgcg gatcctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
1921 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
1981 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
2041 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
2101 gcgaaaatcc tgtttgatgg tggttgacgg cgggatataa catgagctgt cttcggtatc
2161 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
2221 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
2281 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
2341 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
2401 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
2461 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
2521 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
2581 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
2641 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
2701 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
2761 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
2821 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
2881 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
2941 accggcatac tctgcgacat cgtataacgt tactggtttc atcaaaatcg tctccctccg
3001 tttgaatatt tgattgatcg taaccagatg aagcactctt tccactatcc ctacagtgtt
3061 atggcttgaa caatcacgaa acaataattg gtacgtacga tctttcagcc gactcaaaca
3121 tcaaatctta caaatgtagt ctttgaaagt attacatatg taagatttaa atgcaaccgt
3181 tttttcggaa ggaaatgatg acctcgtttc caccggaatt agcttgcatg cctacccaat
3241 cagtacgtta attttggctt taatgagttg taattcctct ggggcaactg ttcctttctt
3301 cgttgctcct cttgcccgcc aggcgatact ttttacctga tcagctaacg ctacgccatc
3361 acgttcctga ccggataaaa caacttcgaa cggatatcct tttgattgcg ttgtacaagg
3421 aacacacaga cacatacctg ttttgttgtt gtacatgaaa ggactcagga caacagctgg
3481 acgatgtcca gcttgctcgc taccttttgt cgggtcaaaa tcaacccaaa tcagatcgcc
3541 catatcgggt acgtatcggc ttaccatacc tgcttcctcc ttaagcttaa ttgttatccg
3601 ctcacaattc cacacattat gccacacctt gtagataaag tcaacaactt tttgcaaaat
3661 ttttcaggaa ttttagcaga ggttgttctg gatgtagaac aaaacatctt tccgctcttg
3721 tgctgttagg atatctttct tggaagctag gtaggcaagg gctacctcaa ataaatcttc
3781 ttcagggtgc gctattttta aggtgcctag tgaggtcttg accacttcac ccataatttc
3841 agtgccgaat agtctggact gggctgtgta gaattcctgc agccctggcg aatggcgatt
3901 ttcgttcgtg aatacatgtt ataataacta taactaataa cgtaacgtga ctggcaagag
3961 atatttttaa aacaatgaat aggtttacac ttactttagt tttatggaaa tgaaagatca
4021 tatcatatat aatctagaat aaaattaact aaaataatta ttatctagat aaaaaattta
4081 gaagccaatg aaatctataa ataaactaaa ttaagtttat ttaattaaca actatggata
4141 taaaataggt actaatcaaa atagtgagga ggatatattt gaatacatac gaacaaatta
4201 ataaagtgaa aaaaatactt cggaaacatt taaaaaataa ccttattggt acttacatgt
4261 ttggatcagg agttgagagt ggactaaaac caaatagtga tcttgacttt ttagtcgtcg
4321 tatctgaacc attgacagat caaagtaaag aaatacttat acaaaaaatt agacctattt
4381 caaaaaaaat aggagataaa agcaacttac gatatattga attaacaatt attattcagc
4441 aagaaatggt accgtggaat catcctccca aacaagaatt tatttatgga gaatggttac
4501 aagagcttta tgaacaagga tacattcctc agaaggaatt aaattcagat ttaaccataa
4561 tgctttacca agcaaaacga aaaaataaaa gaatatacgg aaattatgac ttagaggaat
4621 tactacctga tattccattt tctgatgtga gaagagccat tatggattcg tcagaggaat
4681 taatagataa ttatcaggat gatgaaacca actctatatt aactttatgc cgtatgattt
4741 taactatgga cacgggtaaa atcataccaa aagatattgc gggaaatgca gtggctgaat
4801 cttctccatt agaacatagg gagagaattt tgttagcagt tcgtagttat cttggagaga
4861 atattgaatg gactaatgaa aatgtaaatt taactataaa ctatttaaat aacagattaa
4921 aaaaattata aaaaaattga aaaaatggtg gaaacacttt tttcaatttt tttgttttat
4981 tatttaatat ttgggaaata ttcattctaa ttggtaatca gattttagaa aacaataaac
5041 ccttgcatag ggggatctcg acatggatga gcgatgatga tatccgttta ggctgggcgg
5101 tgatagcttc tcgttcaggc agtacgcctc ttttcttttc cagacctgag ggaggcggaa
5161 atggtgtgag gttcccgggc cggccaattg atgcatcagt tcaaaaggga ttatggtcga
5221 taaagatttt tatctcgtat atatccagtc gaaacctgat ccgtattcac ctggattggc
5281 aatggatgaa accggccaga attccggccg caactggcag tatatagatg gaaagtggca
5341 gccaggtgac aaagcggatg gcaactatat gattcgcgca ttagttgatt atgaagctgc
5401 tgtacctgag attacttcac cgacagacaa atcatacaca aataaggata gcgtcactgt
5461 aaaaggaaac gcttctcctg gcacaacggt acacatttat aatggagaga aagaagcagg
5521 agaaacgaaa gctgctgcgg atggcacgtt ccatgcaggc atcatactca acaagggtga
5581 aaatgagctg acggcaactg catcaactga caacggaaca acagatgcct ccagcccaat
5641 cacggtcacg cttgatcaag aaaagcctga attaacactg gacaatccaa aggatggcgg
5701 gaaaacaaat aaagaaacgc tgactgtcaa aggggctgta tccgatgaca atctgaaaga
5761 cgtcaaggtg aatggcaaaa aagcaacagt agctgatggt tcatactcag cccgtattct
5821 tttggaaaat ggaagaaatg aaatcaaggt aattgctaca gacttggcag gcaacaaaac
5881 gacgaaaaag acagtcattg atgtgaactt tgacaagcct gtcatttccg gcttaattcc
5941 gggagaggat aaaaacttaa aagccggtga atctgtgaaa atcgctttct caagcgctga
6001 ggatttagat gcaacgttta ccattcgtat gccgctgacc aatgcaagag cgagtgtgca
6061 aaatgccacc gaactcccgt taagagaaat ctctccgggg agatatgaag gctattggac
6121 tgccacttct tctattaaag caaaaggagc aaaagtagaa gtgatcgtcc gagatgatta
6181 tggaaatgaa acaagaaaaa ctgcgaatgg aaaacttaat atgaacacag aaaataccgg
6241 tcctagggcg atcgcactag tcgataacca gaagcaataa aaaatcaaat cggatttcac
6301 tatataatct cactttatct aagatgaatc cgatggaagc atcctgtttt ctctcaattt
6361 ttttatctaa aacccagcgt tcgatgcttc tttgagcgaa cgatcaaaaa taagtgcctt
6421 cccatcaaaa aaatattctc aacataaaaa actttgtgta atacttgtaa cgctacatgg
6481 agattaactc aatctagagg gtattaataa tgatccacag tagcgtaaag cgttggggaa
6541 attcaccggc ggtgcggatt ccggctacgt taatgcaggc gctcaatctg aatattgatg
6601 atgaagtgaa gattgacctg gtggatggca aattaattat tgagccagtg cgtaaagagc
6661 ccgtatttac gcttgctgaa ctggtcaacg acatcacgcc ggaaaacctc cacgagaata
6721 tcgactgggg agagccgaaa gataaggaag tctggtaaga gctccagctt ttgttccctt
6781 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
6841 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
6901 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
6961 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
7021 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
7081 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
7141 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
7201 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
7261 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
7321 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
7381 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
7441 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
7501 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
7561 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
7621 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
7681 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
7741 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
7801 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
7861 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
7921 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
7981 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
8041 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
8101 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
8161 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
8221 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
8281 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
8341 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
8401 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
8461 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
8521 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
8581 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
8641 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
8701 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
8761 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
8821 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
8881 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
8941 cgcacatttc cccgaaaagt gccac
//
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
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pIEFBPR枯草分泌质粒
￥100 - 1000
