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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pBI121-EGFP-2
- 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：pBI121-EGFP②植物表达质粒
别称: pBI121-EGFP-2
质粒属性
质粒简介
pBI121-EGFP②质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
OCUS Exported 13629 bp ds-DNA circular SYN 27-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13629)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 27, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..13629
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/label=TetR
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
promoter 2602..2817
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Neo
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 5461..5806
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 5838..6554
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 6561..6578
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
terminator 6598..6850
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 7493..7517
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(9113..10261)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(10560..11354)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 11546..12313
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 12936..18
/label=oriV
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 ggatccccgg gggtaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
5881 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
5941 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
6001 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
6061 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
6121 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
6181 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
6241 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
6301 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
6361 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
6421 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
6481 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
6541 acgagctgta caagctcgag caccaccacc accaccactg agagctcgaa tttccccgat
6601 cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg
6661 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg
6721 acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg
6781 atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg
6841 ttactagatc gggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
6901 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
6961 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg cccgctcctt
7021 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
7081 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
7141 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
7201 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
7261 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa
7321 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
7381 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccacccc
7441 agtacattaa aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac
7501 cacaatatat cctgccacca gccagccaac agctccccga ccggcagctc ggcacaaaat
7561 caccactcga tacaggcagc ccatcagtcc gggacggcgt cagcgggaga gccgttgtaa
7621 ggcggcagac tttgctcatg ttaccgatgc tattcggaag aacggcaact aagctgccgg
7681 gtttgaaaca cggatgatct cgcggagggt agcatgttga ttgtaacgat gacagagcgt
7741 tgctgcctgt gatcaaatat catctccctc gcagagatcc gaattatcag ccttcttatt
7801 catttctcgc ttaaccgtga caggctgtcg atcttgagaa ctatgccgac ataataggaa
7861 atcgctggat aaagccgctg aggaagctga gtggcgctat ttctttagaa gtgaacgttg
7921 acgatatcaa ctcccctatc cattgctcac cgaatggtac aggtcgggga cccgaagttc
7981 cgactgtcgg cctgatgcat ccccggctga tcgaccccag atctggggct gagaaagccc
8041 agtaaggaaa caactgtagg ttcgagtcgc gagatccccc ggaaccaaag gaagtaggtt
8101 aaacccgctc cgatcaggcc gagccacgcc aggccgagaa cattggttcc tgtaggcatc
8161 gggattggcg gatcaaacac taaagctact ggaacgagca gaagtcctcc ggccgccagt
8221 tgccaggcgg taaaggtgag cagaggcacg ggaggttgcc acttgcgggt cagcacggtt
8281 ccgaacgcca tggaaaccgc ccccgccagg cccgctgcga cgccgacagg atctagcgct
8341 gcgtttggtg tcaacaccaa cagcgccacg cccgcagttc cgcaaatagc ccccaggacc
8401 gccatcaatc gtatcgggct acctagcaga gcggcagaga tgaacacgac catcagcggc
8461 tgcacagcgc ctaccgtcgc cgcgaccccg cccggcaggc ggtagaccga aataaacaac
8521 aagctccaga atagcgaaat attaagtgcg ccgaggatga agatgcgcat ccaccagatt
8581 cccgttggaa tctgtcggac gatcatcacg agcaataaac ccgccggcaa cgcccgcagc
8641 agcataccgg cgacccctcg gcctcgctgt tcgggctcca cgaaaacgcc ggacagatgc
8701 gccttgtgag cgtccttggg gccgtcctcc tgtttgaaga ccgacagccc aatgatctcg
8761 ccgtcgatgt aggcgccgaa tgccacggca tctcgcaacc gttcagcgaa cgcctccatg
8821 ggctttttct cctcgtgctc gtaaacggac ccgaacatct ctggagcttt cttcagggcc
8881 gacaatcgga tctcgcggaa atcctgcacg tcggccgctc caagccgtcg aatctgagcc
8941 ttaatcacaa ttgtcaattt taatcctctg tttatcggca gttcgtagag cgcgccgtgc
9001 gtcccgagcg atactgagcg aagcaagtgc gtcgagcagt gcccgcttgt tcctgaaatg
9061 ccagtaaagc gctggctgct gaacccccag ccggaactga ccccacaagg ccctagcgtt
9121 tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg cctcgcaact
9181 cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc
9241 acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt
9301 cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg aatttcgtgt
9361 agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg caacgggacg
9421 ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc
9481 caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg
9541 ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg
9601 tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc tgctgccaca
9661 ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc acgtccttgt
9721 tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg
9781 tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg
9841 caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg
9901 cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt tttcgcttct
9961 tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct
10021 gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca gggggagcca
10081 gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gaccatcgag ccgacggact
10141 ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg
10201 cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac gtccgattca
10261 ttcaccctcc ttgcgggatt gccccgactc acgccggggc aatgtgccct tattcctgat
10321 ttgacccgcc tggtgccttg gtgtccagat aatccacctt atcggcaatg aagtcggtcc
10381 cgtagaccgt ctggccgtcc ttctcgtact tggtattccg aatcttgccc tgcacgaata
10441 ccagcgaccc cttgcccaaa tacttgccgt gggcctcggc ctgagagcca aaacacttga
10501 tgcggaagaa gtcggtgcgc tcctgcttgt cgccggcatc gttgcgccac atctaggtac
10561 taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc
10621 agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg
10681 acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag
10741 ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag
10801 acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta
10861 aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag
10921 taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg
10981 atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt
11041 tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc
11101 cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat
11161 agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc
11221 atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt
11281 ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt
11341 ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag
11401 aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat
11461 accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg
11521 agccgatttt gaaaccacaa ttatgggtga tgctgccaac ttactgattt agtgtatgat
11581 ggtgtttttg aggtgctcca gtggcttctg tgtctatcag ctgtccctcc tgttcagcta
11641 ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctat ctctgctctc
11701 actgccgtaa aacatggcaa ctgcagttca cttacaccgc ttctcaaccc ggtacgcacc
11761 agaaaatcat tgatatggcc atgaatggcg ttggatgccg ggcaacagcc cgcattatgg
11821 gcgttggcct caacacgatt ttacgtcact taaaaaactc aggccgcagt cggtaacctc
11881 gcgcatacag ccgggcagtg acgtcatcgt ctgcgcggaa atggacgaac agtggggcta
11941 tgtcggggct aaatcgcgcc agcgctggct gttttacgcg tatgacagtc tccggaagac
12001 ggttgttgcg cacgtattcg gtgaacgcac tatggcgacg ctggggcgtc ttatgagcct
12061 gctgtcaccc tttgacgtgg tgatatggat gacggatggc tggccgctgt atgaatcccg
12121 cctgaaggga aagctgcacg taatcagcaa gcgatatacg cagcgaattg agcggcataa
12181 cctgaatctg aggcagcacc tggcacggct gggacggaag tcgctgtcgt tctcaaaatc
12241 ggtggagctg catgacaaag tcatcgggca ttatctgaac ataaaacact atcaataagt
12301 tggagtcatt acccaattat gatagaattt acaagctata aggttattgt cctgggtttc
12361 aagcattagt ccatgcaagt ttttatgctt tgcccattct atagatatat tgataagcgc
12421 gctgcctatg ccttgccccc tgaaatcctt acatacggcg atatcttcta tataaaagat
12481 atattatctt atcagtattg tcaatatatt caaggcaatc tgcctcctca tcctcttcat
12541 cctcttcgtc ttggtagctt tttaaatatg gcgcttcata gagtaattct gtaaaggtcc
12601 aattctcgtt ttcatacctc ggtataatct tacctatcac ctcaaatggt tcgctgggtt
12661 tatcgcaccc ccgaacacga gcacggcacc cgcgaccact atgccaagaa tgcccaaggt
12721 aaaaattgcc ggccccgcca tgaagtccgt gaatgccccg acggccgaag tgaagggcag
12781 gccgccaccc aggccgccgc cctcactgcc cggcacctgg tcgctgaatg tcgatgccag
12841 cacctgcggc acgtcaatgc ttccgggcgt cgcgctcggg ctgatcgccc atcccgttac
12901 tgccccgatc ccggcaatgg caaggactgc cagcgctgcc atttttgggg tgaggccgtt
12961 cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg
13021 ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct
13081 ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt
13141 ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct
13201 caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc
13261 gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc
13321 ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt
13381 gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg
13441 ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg
13501 ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca cacggcttcg
13561 acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa
13621 ccagcccgg
//
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：pBI121-EGFP②植物表达质粒
别称: pBI121-EGFP-2
| 启动子: | 35s |
|---|---|
| 复制子: | oriV |
| 原核抗性: | Kan |
| 筛选标记: | G418 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Kan |
| 筛选标记： | Neo/G418 |
| 荧光蛋白： | 绿色 |
质粒简介
pBI121-EGFP②质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
OCUS Exported 13629 bp ds-DNA circular SYN 27-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13629)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 27, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..13629
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/label=TetR
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
promoter 2602..2817
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Neo
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 5461..5806
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 5838..6554
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 6561..6578
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
terminator 6598..6850
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 7493..7517
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(9113..10261)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(10560..11354)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 11546..12313
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 12936..18
/label=oriV
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 ggatccccgg gggtaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
5881 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
5941 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
6001 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
6061 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
6121 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
6181 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
6241 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
6301 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
6361 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
6421 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
6481 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
6541 acgagctgta caagctcgag caccaccacc accaccactg agagctcgaa tttccccgat
6601 cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg
6661 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg
6721 acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg
6781 atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg
6841 ttactagatc gggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
6901 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
6961 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg cccgctcctt
7021 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
7081 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
7141 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
7201 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
7261 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa
7321 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
7381 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccacccc
7441 agtacattaa aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac
7501 cacaatatat cctgccacca gccagccaac agctccccga ccggcagctc ggcacaaaat
7561 caccactcga tacaggcagc ccatcagtcc gggacggcgt cagcgggaga gccgttgtaa
7621 ggcggcagac tttgctcatg ttaccgatgc tattcggaag aacggcaact aagctgccgg
7681 gtttgaaaca cggatgatct cgcggagggt agcatgttga ttgtaacgat gacagagcgt
7741 tgctgcctgt gatcaaatat catctccctc gcagagatcc gaattatcag ccttcttatt
7801 catttctcgc ttaaccgtga caggctgtcg atcttgagaa ctatgccgac ataataggaa
7861 atcgctggat aaagccgctg aggaagctga gtggcgctat ttctttagaa gtgaacgttg
7921 acgatatcaa ctcccctatc cattgctcac cgaatggtac aggtcgggga cccgaagttc
7981 cgactgtcgg cctgatgcat ccccggctga tcgaccccag atctggggct gagaaagccc
8041 agtaaggaaa caactgtagg ttcgagtcgc gagatccccc ggaaccaaag gaagtaggtt
8101 aaacccgctc cgatcaggcc gagccacgcc aggccgagaa cattggttcc tgtaggcatc
8161 gggattggcg gatcaaacac taaagctact ggaacgagca gaagtcctcc ggccgccagt
8221 tgccaggcgg taaaggtgag cagaggcacg ggaggttgcc acttgcgggt cagcacggtt
8281 ccgaacgcca tggaaaccgc ccccgccagg cccgctgcga cgccgacagg atctagcgct
8341 gcgtttggtg tcaacaccaa cagcgccacg cccgcagttc cgcaaatagc ccccaggacc
8401 gccatcaatc gtatcgggct acctagcaga gcggcagaga tgaacacgac catcagcggc
8461 tgcacagcgc ctaccgtcgc cgcgaccccg cccggcaggc ggtagaccga aataaacaac
8521 aagctccaga atagcgaaat attaagtgcg ccgaggatga agatgcgcat ccaccagatt
8581 cccgttggaa tctgtcggac gatcatcacg agcaataaac ccgccggcaa cgcccgcagc
8641 agcataccgg cgacccctcg gcctcgctgt tcgggctcca cgaaaacgcc ggacagatgc
8701 gccttgtgag cgtccttggg gccgtcctcc tgtttgaaga ccgacagccc aatgatctcg
8761 ccgtcgatgt aggcgccgaa tgccacggca tctcgcaacc gttcagcgaa cgcctccatg
8821 ggctttttct cctcgtgctc gtaaacggac ccgaacatct ctggagcttt cttcagggcc
8881 gacaatcgga tctcgcggaa atcctgcacg tcggccgctc caagccgtcg aatctgagcc
8941 ttaatcacaa ttgtcaattt taatcctctg tttatcggca gttcgtagag cgcgccgtgc
9001 gtcccgagcg atactgagcg aagcaagtgc gtcgagcagt gcccgcttgt tcctgaaatg
9061 ccagtaaagc gctggctgct gaacccccag ccggaactga ccccacaagg ccctagcgtt
9121 tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg cctcgcaact
9181 cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc
9241 acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt
9301 cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg aatttcgtgt
9361 agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg caacgggacg
9421 ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc
9481 caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg
9541 ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg
9601 tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc tgctgccaca
9661 ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc acgtccttgt
9721 tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg
9781 tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg
9841 caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg
9901 cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt tttcgcttct
9961 tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct
10021 gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca gggggagcca
10081 gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gaccatcgag ccgacggact
10141 ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg
10201 cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac gtccgattca
10261 ttcaccctcc ttgcgggatt gccccgactc acgccggggc aatgtgccct tattcctgat
10321 ttgacccgcc tggtgccttg gtgtccagat aatccacctt atcggcaatg aagtcggtcc
10381 cgtagaccgt ctggccgtcc ttctcgtact tggtattccg aatcttgccc tgcacgaata
10441 ccagcgaccc cttgcccaaa tacttgccgt gggcctcggc ctgagagcca aaacacttga
10501 tgcggaagaa gtcggtgcgc tcctgcttgt cgccggcatc gttgcgccac atctaggtac
10561 taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc
10621 agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg
10681 acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag
10741 ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag
10801 acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta
10861 aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag
10921 taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg
10981 atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt
11041 tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc
11101 cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat
11161 agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc
11221 atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt
11281 ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt
11341 ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag
11401 aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat
11461 accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg
11521 agccgatttt gaaaccacaa ttatgggtga tgctgccaac ttactgattt agtgtatgat
11581 ggtgtttttg aggtgctcca gtggcttctg tgtctatcag ctgtccctcc tgttcagcta
11641 ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctat ctctgctctc
11701 actgccgtaa aacatggcaa ctgcagttca cttacaccgc ttctcaaccc ggtacgcacc
11761 agaaaatcat tgatatggcc atgaatggcg ttggatgccg ggcaacagcc cgcattatgg
11821 gcgttggcct caacacgatt ttacgtcact taaaaaactc aggccgcagt cggtaacctc
11881 gcgcatacag ccgggcagtg acgtcatcgt ctgcgcggaa atggacgaac agtggggcta
11941 tgtcggggct aaatcgcgcc agcgctggct gttttacgcg tatgacagtc tccggaagac
12001 ggttgttgcg cacgtattcg gtgaacgcac tatggcgacg ctggggcgtc ttatgagcct
12061 gctgtcaccc tttgacgtgg tgatatggat gacggatggc tggccgctgt atgaatcccg
12121 cctgaaggga aagctgcacg taatcagcaa gcgatatacg cagcgaattg agcggcataa
12181 cctgaatctg aggcagcacc tggcacggct gggacggaag tcgctgtcgt tctcaaaatc
12241 ggtggagctg catgacaaag tcatcgggca ttatctgaac ataaaacact atcaataagt
12301 tggagtcatt acccaattat gatagaattt acaagctata aggttattgt cctgggtttc
12361 aagcattagt ccatgcaagt ttttatgctt tgcccattct atagatatat tgataagcgc
12421 gctgcctatg ccttgccccc tgaaatcctt acatacggcg atatcttcta tataaaagat
12481 atattatctt atcagtattg tcaatatatt caaggcaatc tgcctcctca tcctcttcat
12541 cctcttcgtc ttggtagctt tttaaatatg gcgcttcata gagtaattct gtaaaggtcc
12601 aattctcgtt ttcatacctc ggtataatct tacctatcac ctcaaatggt tcgctgggtt
12661 tatcgcaccc ccgaacacga gcacggcacc cgcgaccact atgccaagaa tgcccaaggt
12721 aaaaattgcc ggccccgcca tgaagtccgt gaatgccccg acggccgaag tgaagggcag
12781 gccgccaccc aggccgccgc cctcactgcc cggcacctgg tcgctgaatg tcgatgccag
12841 cacctgcggc acgtcaatgc ttccgggcgt cgcgctcggg ctgatcgccc atcccgttac
12901 tgccccgatc ccggcaatgg caaggactgc cagcgctgcc atttttgggg tgaggccgtt
12961 cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg
13021 ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct
13081 ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt
13141 ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct
13201 caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc
13261 gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc
13321 ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt
13381 gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg
13441 ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg
13501 ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca cacggcttcg
13561 acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa
13621 ccagcccgg
//
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
| P2466/pENTR223-TAP2-1395A人源基因质粒 |
| P2467/pENTR223-OXR1-G3T人源基因质粒 |
| P2468/pENTR223-CFB人源基因质粒 |
| P2469/pENTR223-TTC14人源基因质粒 |
| P2470/pENTR223-USHBP1人源基因质粒 |
| P2471/pENTR223-TCEB3-C2311人源基因质粒 |
| P2472/pENTR223-ATG7人源基因质粒 |
| P2473/pENTR223-C1orf107-T2251G人源基因质粒 |
| P2474/pENTR223-CPT1A人源基因质粒 |
| P2475/pENTR223-YY1AP1人源基因质粒 |
| P2476/pENTR223-PFKM人源基因质粒 |
| P2477/pENTR223-ZNF432-A1943人源基因质粒 |
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文献和实验相关实验
。 GhVP 基因的亚细胞定位 将构建好的 pBI121-GhVP∷GFP 载体用金粉包裹，把金粉包裹好的质粒加入基因枪 GDS-80 轰击洋葱内表皮细胞， 在激光共聚焦显微镜下观察。可以看出，空载体 pBI121-GFP 转化洋葱表皮后， 在整个洋葱细胞均能看到绿色荧光。pBI121-GhVP∷GFP 转化洋葱后，VP 蛋白定位于洋葱表皮细胞的细胞膜上，在玻片上加蔗糖，待质壁分离后，GhVP 蛋白在洋葱表皮细胞的细胞膜和细胞间质中均有分布（图3）。结果表明 GhVP 蛋白是 1 个跨膜蛋白， 与细胞
121 在文献"香蕉 Maasr1 基因 RNAi 植物表达载体的构建及鉴定.热带作物学 报， 2009 年， 30 (3) :333-337. "中公开过，公众可从中国农业科学院棉花研究所抗逆鉴定课 题组实验室获得。基因枪为美国 WEALTEC 的基因枪 GDS-80 。非抗虫棉花材料沪 749513 在文献"高频体细胞胚胎发生的优异棉花种质材料筛选.分子植物育种， 2012 ，10 (6) ，683-688. "中公开过，公众可从中国农业科学院棉花研究 所抗逆鉴定课题组实验室获得。 实施例
pROKII-RNAi 植物质粒 Kan 植物RNAI沉默载体 pBI121 植物质粒 Kan 植物表达载体 pBI121+EGFP 植物质粒 Kan pCAMIBA0380 植物
pBI121-EGFP②植物表达质粒
￥100 - 1000
