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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：
pBE2R-EGFP
- 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：pBE2R-EGFP枯草分泌质粒
别称: pBE2R-EGFP
质粒属性
质粒简介
pBE2R-EGFP是一个枯草杆菌荧光分泌表达质粒。
质粒图谱
质粒序列
LOCUS Exported 7713 bp ds-DNA circular SYN 29-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7713)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 29, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7713
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 419..723
/label=P43
misc_feature 756..1088
/label=Apr sp+pro
CDS 1095..1811
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1818..1835
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 2652..2756
/gene="bla"
/label=AmpR promoter
CDS 2757..3617
/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"
rep_origin 3788..4376
/direction=RIGHT
/label=ColE1 ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5779..6549)
/codon_start=1
/label=Kan
/translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHG
QFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTT
FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQL-SD
SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
CDS complement(6709..7713)
/codon_start=1
/gene="repB"
/product="RepB replication protein"
/label=repB
/note="from Enterococcus faecalis plasmid pAM-alpha-1"
/db_xref="GI:22652809"
/protein_id="AAN03827.1"
/translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSL-SDM-AQGFRRMMQYKKINKNLV
GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRL-SDLEEGLHRKRLIS
YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
ORIGIN
1 ctatttagtt atttgtttag tcacttataa ctttaacaga tggggttttt ctgtgcaacc
61 aattttaagg gttttcaata ctttaaaaca catacatacc aacacttcaa cgcacctttc
121 agcaactaaa ataaaaatga cgttatttct atatgtatca agataagaaa gaacaagttc
181 aaaaccatca aaaaaagaca ccttttcagg tgcttttttt attttataaa ctcattccct
241 gatctcgact tcgttctttt tttacctctc ggttatgagt tagttcaaat tcgttctttt
301 taggttctaa atcgtgtttt tcttggaatt gtgctgtttt atcctttacc ttgtctacaa
361 accccttaaa aacgttttta aaggctttta agccgtctgt acgttcctta aggaattcga
421 gctcagcttt attgagtgga tgattatatt ccttttgata ggtggtatgt tttcgcttga
481 acttttaaat acagccattg aacatacggt tgatttaata actgacaaac atcaccctct
541 tgctaaagcg gccaaggacg ctgccgccgg ggctgtttgc gtttttgccg tgatttcgtg
601 tattattggt ttacttattt ttttgccaaa gctgtaatgg ctgaaaattc ttacatttat
661 tttacatttt tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat ataaagtgat
721 agcggtacca ttataggtaa gagaggaatg tacacatgaa gaaaccgttg gggaaaattg
781 tcgcaagcac cgcactactc atttctgttg cttttagttc atcgatcgca tcggctgctg
841 aagaagcaaa agaaaaatat ttaattggct ttaatgagca ggaagctgtc agtgagtttg
901 tagaacaagt agaggcaaat gacgaggtcg ccattctctc tgaggaagag gaagtcgaaa
961 ttgaattgct tcatgaattt gaaacgattc ctgttttatc cgttgagtta agcccagaag
1021 atgtggacgc gcttgaactc gatccagcga tttcttatat tgaagaggat gcagaagtaa
1081 cgacaatggg atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
1141 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
1201 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
1261 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
1321 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
1381 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
1441 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
1501 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
1561 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
1621 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
1681 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
1741 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
1801 agctgtacaa gctcgagcac caccaccacc accacaagct tgtattctat atagtgtcac
1861 ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg ttctaacgac
1921 aatatgtaca agcctaattg tgtagcatct ggcttactga agcagaccct atcatctctc
1981 tcgtaaactg ccgtcagagt cggtttggtt ggacgaacct tctgagtttc tggtaacgcc
2041 gttccgcacc ccggaaatgg tcagcgaacc aatcagcagg gtcatcgcta gccagatcct
2101 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
2161 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
2221 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
2281 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
2341 atgcaggagt cgcataaggg agagcgtcga tatggtgcac tctcagtaca atctgctctg
2401 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
2461 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
2521 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
2581 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
2641 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
2701 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
2761 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
2821 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
2881 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
2941 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
3001 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
3061 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
3121 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
3181 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
3241 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
3301 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
3361 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
3421 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
3481 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
3541 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
3601 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
3661 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
3721 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
3781 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
3841 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
3901 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
3961 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
4021 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
4081 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
4141 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
4201 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
4261 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
4321 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
4381 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
4441 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
4501 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
4561 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgatttc
4621 actttttgca ttctacaaac tgcataactc atatgtaaat cgctcctttt taggtggcac
4681 aaatgtgagg cattttcgct ctttccggca accacttcca agtaaagtat aacacactat
4741 actttatatt cataaagtgt gtgctctgcg aggctgtcgg cagtgccgac caaaaccata
4801 aaacctttaa gacctttctt ttttttacga gaaaaaagaa acaaaaaaac ctgccctctg
4861 ccacctcagc aaaggggggt tttgctctcg tgctcgttta aaaatcagca agggacaggt
4921 agtatttttt gagaagatca ctcaaaaaat ctccaccttt aaacccttgc caatttttat
4981 tttgtccgtt ttgtctagct taccgaaagc cagactcagc aagaataaaa tttttattgt
5041 ctttcggttt tctagtgtaa cggacaaaac cactcaaaat aaaaaagata caagagaggt
5101 ctctcgtatc ttttattcag caatcgcgcc cgattgctga acagattaat aatagatttt
5161 agctttttat ttgttgaaaa aagctaatca aattgttgtc gggatcaatt actgcaaagt
5221 ctcgttcatc ccaccactga tcttttaatg atgtattggg gtgcaaaatg cccaaaggct
5281 taatatgttg atataattca tcaattccct ctacttcaat gcggcaacta gcagtaccag
5341 caataaacga ctccgcacct gtacaaaccg gtgaatcatt actacgagag cgccagcttc
5401 atcacttgcc tcccatagat gaatccgaac ctcattacac attagaactg cgaatccatc
5461 ttcatggtga accaaagtga aacctagttt atcgcaataa aaacctatac tctttttaat
5521 atccccgact ggcaatgccg ggatagactg taacattctc acgcataaaa tcccctttca
5581 ttttctaatg taaatctatt accttattat taattcaatt cgctcataat taatcctttt
5641 tcttattacg caaaatggcc cgatttaagc acacccttta ttccgttaat gcgccatgac
5701 agccatgata attactaata ctaggagaag ttaataaata cgtaaccaac atgattaaca
5761 attattagag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg
5821 tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc
5881 agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag
5941 atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg
6001 atgcagacca atcaacatgg cacctgccat tgctacctgt acagtcaagg atggtagaaa
6061 tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc
6121 tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg
6181 atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc
6241 atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct
6301 atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc
6361 ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc
6421 aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc
6481 cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac
6541 tattctcatt cccttttcag ataattttag atttgctttt ctaaataaga atatttggag
6601 agcaccgttc ttattcagct attaataact cgtcttccta agcatccttc aatcctttta
6661 ataacaatta tagcatctaa tcttcaacaa actggcccgt ttgttgaact actctttaat
6721 aaaataattt ttccgttccc aattccacat tgcaataata gaaaatccat cttcatcggc
6781 tttttcgtca tcatctgtat gaatcaaatc gccttcttct gtgtcatcaa ggtttaattt
6841 tttatgtatt tcttttaaca aaccaccata ggagattaac cttttacggt gtaaaccttc
6901 ctccaaatca gacaaacgtt tcaaattctt ttcttcatca tcggtcataa aatccgtatc
6961 ctttacagga tattttgcag tttcgtcaat tgccgattgt atatccgatt tatatttatt
7021 tttcggtcga atcatttgaa cttttacatt tggatcatag tctaatttca ttgccttttt
7081 ccaaaattga atccattgtt tttgattcac gtagttttct gtattcttaa aataagttgg
7141 ttccacacat accaatacat gcatgtgctg attataagaa ttatctttat tatttattgt
7201 cacttccgtt gcacgcataa aaccaacaag atttttatta atttttttat attgcatcat
7261 tcggcgaaat ccttgagcca tatctgacaa actcttattt aattcttcgc catcataaac
7321 atttttaact gttaatgtga gaaacaacca acgaactgtt ggcttttgtt taataacttc
7381 agcaacaacc ttttgtgact gaatgccatg tttcattgct ctcctccagt tgcacattgg
7441 acaaagcctg gatttacaaa accacactcg atacaacttt ctttcgcctg tttcacgatt
7501 ttgtttatac tctaatattt cagcacaatc ttttactctt tcagcctttt taaattcaag
7561 aatatgcaga agttcaaagt aatcaacatt agcgattttc ttttctctcc atggtctcac
7621 ttttccactt tttgtcttgt ccactaaaac ccttgatttt tcatctgaat aaatgctact
7681 attaggacac ataatattaa aagaaacccc cat
//
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：pBE2R-EGFP枯草分泌质粒
别称: pBE2R-EGFP
| 启动子: | P43 |
|---|---|
| 复制子: | repB |
| 克隆菌株: | Stbl3 |
| 培养条件: | 30度 |
质粒属性
| 载体宿主： | 枯草杆菌 |
|---|---|
| 载体用途： | 蛋白表达,分泌表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | |
| 荧光蛋白： | 绿色 |
质粒简介
pBE2R-EGFP是一个枯草杆菌荧光分泌表达质粒。
质粒图谱
质粒序列
LOCUS Exported 7713 bp ds-DNA circular SYN 29-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7713)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 29, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7713
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 419..723
/label=P43
misc_feature 756..1088
/label=Apr sp+pro
CDS 1095..1811
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1818..1835
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 2652..2756
/gene="bla"
/label=AmpR promoter
CDS 2757..3617
/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"
rep_origin 3788..4376
/direction=RIGHT
/label=ColE1 ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5779..6549)
/codon_start=1
/label=Kan
/translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHG
QFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTT
FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQL-SD
SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
CDS complement(6709..7713)
/codon_start=1
/gene="repB"
/product="RepB replication protein"
/label=repB
/note="from Enterococcus faecalis plasmid pAM-alpha-1"
/db_xref="GI:22652809"
/protein_id="AAN03827.1"
/translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSL-SDM-AQGFRRMMQYKKINKNLV
GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRL-SDLEEGLHRKRLIS
YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
ORIGIN
1 ctatttagtt atttgtttag tcacttataa ctttaacaga tggggttttt ctgtgcaacc
61 aattttaagg gttttcaata ctttaaaaca catacatacc aacacttcaa cgcacctttc
121 agcaactaaa ataaaaatga cgttatttct atatgtatca agataagaaa gaacaagttc
181 aaaaccatca aaaaaagaca ccttttcagg tgcttttttt attttataaa ctcattccct
241 gatctcgact tcgttctttt tttacctctc ggttatgagt tagttcaaat tcgttctttt
301 taggttctaa atcgtgtttt tcttggaatt gtgctgtttt atcctttacc ttgtctacaa
361 accccttaaa aacgttttta aaggctttta agccgtctgt acgttcctta aggaattcga
421 gctcagcttt attgagtgga tgattatatt ccttttgata ggtggtatgt tttcgcttga
481 acttttaaat acagccattg aacatacggt tgatttaata actgacaaac atcaccctct
541 tgctaaagcg gccaaggacg ctgccgccgg ggctgtttgc gtttttgccg tgatttcgtg
601 tattattggt ttacttattt ttttgccaaa gctgtaatgg ctgaaaattc ttacatttat
661 tttacatttt tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat ataaagtgat
721 agcggtacca ttataggtaa gagaggaatg tacacatgaa gaaaccgttg gggaaaattg
781 tcgcaagcac cgcactactc atttctgttg cttttagttc atcgatcgca tcggctgctg
841 aagaagcaaa agaaaaatat ttaattggct ttaatgagca ggaagctgtc agtgagtttg
901 tagaacaagt agaggcaaat gacgaggtcg ccattctctc tgaggaagag gaagtcgaaa
961 ttgaattgct tcatgaattt gaaacgattc ctgttttatc cgttgagtta agcccagaag
1021 atgtggacgc gcttgaactc gatccagcga tttcttatat tgaagaggat gcagaagtaa
1081 cgacaatggg atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
1141 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
1201 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
1261 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
1321 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
1381 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
1441 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
1501 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
1561 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
1621 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
1681 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
1741 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
1801 agctgtacaa gctcgagcac caccaccacc accacaagct tgtattctat atagtgtcac
1861 ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg ttctaacgac
1921 aatatgtaca agcctaattg tgtagcatct ggcttactga agcagaccct atcatctctc
1981 tcgtaaactg ccgtcagagt cggtttggtt ggacgaacct tctgagtttc tggtaacgcc
2041 gttccgcacc ccggaaatgg tcagcgaacc aatcagcagg gtcatcgcta gccagatcct
2101 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
2161 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
2221 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
2281 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
2341 atgcaggagt cgcataaggg agagcgtcga tatggtgcac tctcagtaca atctgctctg
2401 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
2461 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
2521 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
2581 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
2641 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
2701 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
2761 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
2821 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
2881 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
2941 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
3001 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
3061 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
3121 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
3181 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
3241 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
3301 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
3361 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
3421 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
3481 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
3541 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
3601 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
3661 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
3721 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
3781 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
3841 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
3901 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
3961 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
4021 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
4081 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
4141 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
4201 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
4261 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
4321 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
4381 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
4441 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
4501 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
4561 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgatttc
4621 actttttgca ttctacaaac tgcataactc atatgtaaat cgctcctttt taggtggcac
4681 aaatgtgagg cattttcgct ctttccggca accacttcca agtaaagtat aacacactat
4741 actttatatt cataaagtgt gtgctctgcg aggctgtcgg cagtgccgac caaaaccata
4801 aaacctttaa gacctttctt ttttttacga gaaaaaagaa acaaaaaaac ctgccctctg
4861 ccacctcagc aaaggggggt tttgctctcg tgctcgttta aaaatcagca agggacaggt
4921 agtatttttt gagaagatca ctcaaaaaat ctccaccttt aaacccttgc caatttttat
4981 tttgtccgtt ttgtctagct taccgaaagc cagactcagc aagaataaaa tttttattgt
5041 ctttcggttt tctagtgtaa cggacaaaac cactcaaaat aaaaaagata caagagaggt
5101 ctctcgtatc ttttattcag caatcgcgcc cgattgctga acagattaat aatagatttt
5161 agctttttat ttgttgaaaa aagctaatca aattgttgtc gggatcaatt actgcaaagt
5221 ctcgttcatc ccaccactga tcttttaatg atgtattggg gtgcaaaatg cccaaaggct
5281 taatatgttg atataattca tcaattccct ctacttcaat gcggcaacta gcagtaccag
5341 caataaacga ctccgcacct gtacaaaccg gtgaatcatt actacgagag cgccagcttc
5401 atcacttgcc tcccatagat gaatccgaac ctcattacac attagaactg cgaatccatc
5461 ttcatggtga accaaagtga aacctagttt atcgcaataa aaacctatac tctttttaat
5521 atccccgact ggcaatgccg ggatagactg taacattctc acgcataaaa tcccctttca
5581 ttttctaatg taaatctatt accttattat taattcaatt cgctcataat taatcctttt
5641 tcttattacg caaaatggcc cgatttaagc acacccttta ttccgttaat gcgccatgac
5701 agccatgata attactaata ctaggagaag ttaataaata cgtaaccaac atgattaaca
5761 attattagag gtcatcgttc aaaatggtat gcgttttgac acatccacta tatatccgtg
5821 tcgttctgtc cactcctgaa tcccattcca gaaattctct agcgattcca gaagtttctc
5881 agagtcggaa agttgaccag acattacgaa ctggcacaga tggtcataac ctgaaggaag
5941 atctgattgc ttaactgctt cagttaagac cgaagcgctc gtcgtataac agatgcgatg
6001 atgcagacca atcaacatgg cacctgccat tgctacctgt acagtcaagg atggtagaaa
6061 tgttgtcggt ccttgcacac gaatattacg ccatttgcct gcatattcaa acagctcttc
6121 tacgataagg gcacaaatcg catcgtggaa cgtttgggct tctaccgatt tagcagtttg
6181 atacactttc tctaagtatc cacctgaatc ataaatcggc aaaatagaga aaaattgacc
6241 atgtgtaagc ggccaatctg attccacctg agatgcataa tctagtagaa tctcttcgct
6301 atcaaaattc acttccacct tccactcacc ggttgtccat tcatggctga actctgcttc
6361 ctctgttgac atgacacaca tcatctcaat atccgaatag ggcccatcag tctgacgacc
6421 aagagagcca taaacaccaa tagccttaac atcatcccca tatttatcca atattcgttc
6481 cttaatttca tgaacaatct tcattctttc ttctctagtc attattattg gtccattcac
6541 tattctcatt cccttttcag ataattttag atttgctttt ctaaataaga atatttggag
6601 agcaccgttc ttattcagct attaataact cgtcttccta agcatccttc aatcctttta
6661 ataacaatta tagcatctaa tcttcaacaa actggcccgt ttgttgaact actctttaat
6721 aaaataattt ttccgttccc aattccacat tgcaataata gaaaatccat cttcatcggc
6781 tttttcgtca tcatctgtat gaatcaaatc gccttcttct gtgtcatcaa ggtttaattt
6841 tttatgtatt tcttttaaca aaccaccata ggagattaac cttttacggt gtaaaccttc
6901 ctccaaatca gacaaacgtt tcaaattctt ttcttcatca tcggtcataa aatccgtatc
6961 ctttacagga tattttgcag tttcgtcaat tgccgattgt atatccgatt tatatttatt
7021 tttcggtcga atcatttgaa cttttacatt tggatcatag tctaatttca ttgccttttt
7081 ccaaaattga atccattgtt tttgattcac gtagttttct gtattcttaa aataagttgg
7141 ttccacacat accaatacat gcatgtgctg attataagaa ttatctttat tatttattgt
7201 cacttccgtt gcacgcataa aaccaacaag atttttatta atttttttat attgcatcat
7261 tcggcgaaat ccttgagcca tatctgacaa actcttattt aattcttcgc catcataaac
7321 atttttaact gttaatgtga gaaacaacca acgaactgtt ggcttttgtt taataacttc
7381 agcaacaacc ttttgtgact gaatgccatg tttcattgct ctcctccagt tgcacattgg
7441 acaaagcctg gatttacaaa accacactcg atacaacttt ctttcgcctg tttcacgatt
7501 ttgtttatac tctaatattt cagcacaatc ttttactctt tcagcctttt taaattcaag
7561 aatatgcaga agttcaaagt aatcaacatt agcgattttc ttttctctcc atggtctcac
7621 ttttccactt tttgtcttgt ccactaaaac ccttgatttt tcatctgaat aaatgctact
7681 attaggacac ataatattaa aagaaacccc cat
//
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
| P4751/pCMV-SPORT6-PRRT1人源基因质粒 |
| P4752/pCMV-SPORT6-TM4SF19人源基因质粒 |
| P4753/pCMV-SPORT6-MSX2（1点突变）人源基因质粒 |
| P4754/pCMV-SPORT6-SSBP2人源基因质粒 |
| P4755/pCMV-SPORT6-CYB5A人源基因质粒 |
| P4756/pCMV-SPORT6-NME4人源基因质粒 |
| P4757/pCMV-SPORT6-RAB34人源基因质粒 |
| P4758/pCMV-SPORT6-AIDA人源基因质粒 |
| P4759/pCMV-SPORT6-TCIRG1人源基因质粒 |
| P4760/pCMV-SPORT6-RNASE4人源基因质粒 |
| P4761/pCMV-SPORT6-TEAD4人源基因质粒 |
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pBE2R-EGFP枯草分泌质粒
￥100 - 1000
