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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：
pGWB18
- 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：pGWB18植物表达质粒
别称: pGWB18
质粒属性
质粒简介
pGWB18是一个GateWay系列的植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17409 bp ds-DNA circular SYN 27-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..17409
/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 2634..2817
/function="NOS promoter"
/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=NeoR/KanR
/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 5842..5871
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5878..5907
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5923..5952
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5959..5988
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
protein_bind 6007..6131
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 6168..6198
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 6252..6911
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 7253..7558
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(7599..7723)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
terminator 7749..8001
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8430..8682
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(9150..10175)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDA-IAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(10185..10529)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 11273..11297
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12893..14041)
/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(14340..15134)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15326..16093
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16716..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 atgagcgggt taattaacgg tgaacaaaag ctaatctccg aggaagactt gaacggtgaa
5881 caaaaattaa tctcagaaga agacttgaac ggactcgacg gtgaacaaaa gttgatttct
5941 gaagaagatt tgaacggtga acaaaagcta atctccgagg aagacttgaa cggtagcgct
6001 gttatcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
6061 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
6121 cagtcactat ggcggcccct cgaggggccg cattaggcac cccaggcttt acactttatg
6181 cttccggctc gtataatgtg tggattttga gttaggatcc ggcgagattt tcaggagcta
6241 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
6301 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
6361 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
6421 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
6481 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
6541 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
6601 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
6661 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
6721 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
6781 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg
6841 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
6901 gcggggcgta aacgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc
6961 gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa
7021 gaggtgtgct atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct
7081 caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc
7141 cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc
7201 cggtttattg aaatgaacgg ctcttttgct gacgagaaca gggactggtg aaatgcagtt
7261 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
7321 tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
7381 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
7441 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
7501 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
7561 gtcaggctcc cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg
7621 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
7681 tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtgataacc tagagctcga
7741 atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg
7801 gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca
7861 tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca
7921 tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg
7981 tgtcatctat gttactagat cgggaattag cttcatcaac gcaagacatg cgcacgaccg
8041 tctgacagga gaggaatttc cgacgagcac agaaaggact tgctcttgga cgtaggccta
8101 tttctcaggc acatgtatca agtgttcgga cgtgggtttt cgatggtgta tcagccgccg
8161 ccaactggga gatgaggagg ctttcttggg gggcagtcag cagttcattt cacaagacag
8221 aggaacttgt aaggagatgc actgatttat cttggcgcaa accagcagga cgaattagtg
8281 ggaatagccc gcgaatatct aagttatgcc tgtcggcatg agcagaaact tccaattcga
8341 aacagtttgg agaggttgtt tttgggcata ccttttgtta gtcagcctct cgattgctca
8401 tcgtcattac acagtaccga agtttgatcg atctagtaac atagatgaca ccgcgcgcga
8461 taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg
8521 cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat
8581 tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg
8641 attcaatctt aagaaacttt attgccaaat gtttgaacga tctgcttcga cgcactcctt
8701 ctttactcca ccatctcgtc cttattgaaa acgtgggtag caccaaaacg aatcaagtcg
8761 ctggaactga agttaccaat cacgctggat gatttgccag ttggattaat cttgcctttc
8821 cccgcatgaa taatattgat gaatgcatgc gtgaggggta tttcgatttt ggcaatagct
8881 gcaattgccg cgacatcctc caacgagcat aattcttcag aaaaatagcg atgttccatg
8941 ttgtcagggc atgcatgatg cacgttatga ggtgacggtg ctaggcagta ttccctcaaa
9001 gtttcatagt cagtatcata ttcatcattg cattcctgca agagagaatt gagacgcaat
9061 ccacacgctg cggcaacctt ccggcgttcg tggtctattt gctcttggac gttgcaaacg
9121 taagtgttgg atcccggtcg gcatctactc tattcctttg ccctcggacg agtgctgggg
9181 cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt
9241 ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat
9301 cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg
9361 tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc
9421 ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
9481 atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct
9541 gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc
9601 cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac
9661 gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg
9721 catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
9781 ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc
9841 agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg
9901 gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg
9961 agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag
10021 ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
10081 tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc
10141 tcgacagacg tcgcggtgag ttcaggcttt ttcatatcgg ggtcgtcctc tccaaatgaa
10201 atgaacttcc ttatatagag gaagggtctt gcgaaggata gtgggattgt gcgtcatccc
10261 ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt
10321 ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg
10381 catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct
10441 tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc
10501 ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt
10561 tgatattctt ggagtagacg agagtgtcgt gctccaccat gttgacggat ctctaggacg
10621 cgtcctagaa gctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
10681 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
10741 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg cccgctcctt
10801 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
10861 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
10921 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
10981 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
11041 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa
11101 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
11161 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccacccc
11221 agtacattaa aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac
11281 cacaatatat cctgccacca gccagccaac agctccccga ccggcagctc ggcacaaaat
11341 caccactcga tacaggcagc ccatcagtcc gggacggcgt cagcgggaga gccgttgtaa
11401 ggcggcagac tttgctcatg ttaccgatgc tattcggaag aacggcaact aagctgccgg
11461 gtttgaaaca cggatgatct cgcggagggt agcatgttga ttgtaacgat gacagagcgt
11521 tgctgcctgt gatcaaatat catctccctc gcagagatcc gaattatcag ccttcttatt
11581 catttctcgc ttaaccgtga caggctgtcg atcttgagaa ctatgccgac ataataggaa
11641 atcgctggat aaagccgctg aggaagctga gtggcgctat ttctttagaa gtgaacgttg
11701 acgatatcaa ctcccctatc cattgctcac cgaatggtac aggtcgggga cccgaagttc
11761 cgactgtcgg cctgatgcat ccccggctga tcgaccccag atctggggct gagaaagccc
11821 agtaaggaaa caactgtagg ttcgagtcgc gagatccccc ggaaccaaag gaagtaggtt
11881 aaacccgctc cgatcaggcc gagccacgcc aggccgagaa cattggttcc tgtaggcatc
11941 gggattggcg gatcaaacac taaagctact ggaacgagca gaagtcctcc ggccgccagt
12001 tgccaggcgg taaaggtgag cagaggcacg ggaggttgcc acttgcgggt cagcacggtt
12061 ccgaacgcca tggaaaccgc ccccgccagg cccgctgcga cgccgacagg atctagcgct
12121 gcgtttggtg tcaacaccaa cagcgccacg cccgcagttc cgcaaatagc ccccaggacc
12181 gccatcaatc gtatcgggct acctagcaga gcggcagaga tgaacacgac catcagcggc
12241 tgcacagcgc ctaccgtcgc cgcgaccccg cccggcaggc ggtagaccga aataaacaac
12301 aagctccaga atagcgaaat attaagtgcg ccgaggatga agatgcgcat ccaccagatt
12361 cccgttggaa tctgtcggac gatcatcacg agcaataaac ccgccggcaa cgcccgcagc
12421 agcataccgg cgacccctcg gcctcgctgt tcgggctcca cgaaaacgcc ggacagatgc
12481 gccttgtgag cgtccttggg gccgtcctcc tgtttgaaga ccgacagccc aatgatctcg
12541 ccgtcgatgt aggcgccgaa tgccacggca tctcgcaacc gttcagcgaa cgcctccatg
12601 ggctttttct cctcgtgctc gtaaacggac ccgaacatct ctggagcttt cttcagggcc
12661 gacaatcgga tctcgcggaa atcctgcacg tcggccgctc caagccgtcg aatctgagcc
12721 ttaatcacaa ttgtcaattt taatcctctg tttatcggca gttcgtagag cgcgccgtgc
12781 gtcccgagcg atactgagcg aagcaagtgc gtcgagcagt gcccgcttgt tcctgaaatg
12841 ccagtaaagc gctggctgct gaacccccag ccggaactga ccccacaagg ccctagcgtt
12901 tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg cctcgcaact
12961 cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc
13021 acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt
13081 cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg aatttcgtgt
13141 agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg caacgggacg
13201 ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc
13261 caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg
13321 ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg
13381 tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc tgctgccaca
13441 ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc acgtccttgt
13501 tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg
13561 tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg
13621 caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg
13681 cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt tttcgcttct
13741 tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct
13801 gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca gggggagcca
13861 gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gaccatcgag ccgacggact
13921 ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg
13981 cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac gtccgattca
14041 ttcaccctcc ttgcgggatt gccccgactc acgccggggc aatgtgccct tattcctgat
14101 ttgacccgcc tggtgccttg gtgtccagat aatccacctt atcggcaatg aagtcggtcc
14161 cgtagaccgt ctggccgtcc ttctcgtact tggtattccg aatcttgccc tgcacgaata
14221 ccagcgaccc cttgcccaaa tacttgccgt gggcctcggc ctgagagcca aaacacttga
14281 tgcggaagaa gtcggtgcgc tcctgcttgt cgccggcatc gttgcgccac atctaggtac
14341 taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc
14401 agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg
14461 acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag
14521 ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag
14581 acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta
14641 aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag
14701 taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg
14761 atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt
14821 tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc
14881 cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat
14941 agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc
15001 atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt
15061 ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt
15121 ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag
15181 aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat
15241 accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg
15301 agccgatttt gaaaccacaa ttatgggtga tgctgccaac ttactgattt agtgtatgat
15361 ggtgtttttg aggtgctcca gtggcttctg tgtctatcag ctgtccctcc tgttcagcta
15421 ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctat ctctgctctc
15481 actgccgtaa aacatggcaa ctgcagttca cttacaccgc ttctcaaccc ggtacgcacc
15541 agaaaatcat tgatatggcc atgaatggcg ttggatgccg ggcaacagcc cgcattatgg
15601 gcgttggcct caacacgatt ttacgtcact taaaaaactc aggccgcagt cggtaacctc
15661 gcgcatacag ccgggcagtg acgtcatcgt ctgcgcggaa atggacgaac agtggggcta
15721 tgtcggggct aaatcgcgcc agcgctggct gttttacgcg tatgacagtc tccggaagac
15781 ggttgttgcg cacgtattcg gtgaacgcac tatggcgacg ctggggcgtc ttatgagcct
15841 gctgtcaccc tttgacgtgg tgatatggat gacggatggc tggccgctgt atgaatcccg
15901 cctgaaggga aagctgcacg taatcagcaa gcgatatacg cagcgaattg agcggcataa
15961 cctgaatctg aggcagcacc tggcacggct gggacggaag tcgctgtcgt tctcaaaatc
16021 ggtggagctg catgacaaag tcatcgggca ttatctgaac ataaaacact atcaataagt
16081 tggagtcatt acccaattat gatagaattt acaagctata aggttattgt cctgggtttc
16141 aagcattagt ccatgcaagt ttttatgctt tgcccattct atagatatat tgataagcgc
16201 gctgcctatg ccttgccccc tgaaatcctt acatacggcg atatcttcta tataaaagat
16261 atattatctt atcagtattg tcaatatatt caaggcaatc tgcctcctca tcctcttcat
16321 cctcttcgtc ttggtagctt tttaaatatg gcgcttcata gagtaattct gtaaaggtcc
16381 aattctcgtt ttcatacctc ggtataatct tacctatcac ctcaaatggt tcgctgggtt
16441 tatcgcaccc ccgaacacga gcacggcacc cgcgaccact atgccaagaa tgcccaaggt
16501 aaaaattgcc ggccccgcca tgaagtccgt gaatgccccg acggccgaag tgaagggcag
16561 gccgccaccc aggccgccgc cctcactgcc cggcacctgg tcgctgaatg tcgatgccag
16621 cacctgcggc acgtcaatgc ttccgggcgt cgcgctcggg ctgatcgccc atcccgttac
16681 tgccccgatc ccggcaatgg caaggactgc cagcgctgcc atttttgggg tgaggccgtt
16741 cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg
16801 ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct
16861 ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt
16921 ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct
16981 caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc
17041 gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc
17101 ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt
17161 gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg
17221 ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg
17281 ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca cacggcttcg
17341 acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa
17401 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：pGWB18植物表达质粒
别称: pGWB18
| 启动子: | 35s |
|---|---|
| 复制子: | oriV |
| 原核抗性: | Kan |
| 筛选标记: | Hyg,G418 |
| 克隆菌株: | DB3.1 |
| 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： | 空载体 |
| 基因类型： | ORF,GateWay |
| 原核抗性： | Kan |
| 筛选标记： | Neo/G418,Hyg |
| 荧光蛋白： |
质粒简介
pGWB18是一个GateWay系列的植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17409 bp ds-DNA circular SYN 27-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..17409
/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 2634..2817
/function="NOS promoter"
/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=NeoR/KanR
/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 5842..5871
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5878..5907
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5923..5952
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5959..5988
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
protein_bind 6007..6131
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 6168..6198
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 6252..6911
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 7253..7558
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(7599..7723)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
terminator 7749..8001
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8430..8682
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(9150..10175)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDA-IAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(10185..10529)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 11273..11297
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12893..14041)
/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(14340..15134)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15326..16093
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16716..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 atgagcgggt taattaacgg tgaacaaaag ctaatctccg aggaagactt gaacggtgaa
5881 caaaaattaa tctcagaaga agacttgaac ggactcgacg gtgaacaaaa gttgatttct
5941 gaagaagatt tgaacggtga acaaaagcta atctccgagg aagacttgaa cggtagcgct
6001 gttatcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
6061 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
6121 cagtcactat ggcggcccct cgaggggccg cattaggcac cccaggcttt acactttatg
6181 cttccggctc gtataatgtg tggattttga gttaggatcc ggcgagattt tcaggagcta
6241 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
6301 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
6361 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
6421 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
6481 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
6541 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
6601 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
6661 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
6721 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
6781 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg
6841 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
6901 gcggggcgta aacgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc
6961 gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa
7021 gaggtgtgct atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct
7081 caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc
7141 cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc
7201 cggtttattg aaatgaacgg ctcttttgct gacgagaaca gggactggtg aaatgcagtt
7261 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
7321 tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
7381 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
7441 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
7501 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
7561 gtcaggctcc cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg
7621 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
7681 tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtgataacc tagagctcga
7741 atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg
7801 gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca
7861 tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca
7921 tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg
7981 tgtcatctat gttactagat cgggaattag cttcatcaac gcaagacatg cgcacgaccg
8041 tctgacagga gaggaatttc cgacgagcac agaaaggact tgctcttgga cgtaggccta
8101 tttctcaggc acatgtatca agtgttcgga cgtgggtttt cgatggtgta tcagccgccg
8161 ccaactggga gatgaggagg ctttcttggg gggcagtcag cagttcattt cacaagacag
8221 aggaacttgt aaggagatgc actgatttat cttggcgcaa accagcagga cgaattagtg
8281 ggaatagccc gcgaatatct aagttatgcc tgtcggcatg agcagaaact tccaattcga
8341 aacagtttgg agaggttgtt tttgggcata ccttttgtta gtcagcctct cgattgctca
8401 tcgtcattac acagtaccga agtttgatcg atctagtaac atagatgaca ccgcgcgcga
8461 taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg
8521 cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat
8581 tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg
8641 attcaatctt aagaaacttt attgccaaat gtttgaacga tctgcttcga cgcactcctt
8701 ctttactcca ccatctcgtc cttattgaaa acgtgggtag caccaaaacg aatcaagtcg
8761 ctggaactga agttaccaat cacgctggat gatttgccag ttggattaat cttgcctttc
8821 cccgcatgaa taatattgat gaatgcatgc gtgaggggta tttcgatttt ggcaatagct
8881 gcaattgccg cgacatcctc caacgagcat aattcttcag aaaaatagcg atgttccatg
8941 ttgtcagggc atgcatgatg cacgttatga ggtgacggtg ctaggcagta ttccctcaaa
9001 gtttcatagt cagtatcata ttcatcattg cattcctgca agagagaatt gagacgcaat
9061 ccacacgctg cggcaacctt ccggcgttcg tggtctattt gctcttggac gttgcaaacg
9121 taagtgttgg atcccggtcg gcatctactc tattcctttg ccctcggacg agtgctgggg
9181 cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt
9241 ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat
9301 cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg
9361 tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc
9421 ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
9481 atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct
9541 gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc
9601 cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac
9661 gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg
9721 catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
9781 ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc
9841 agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg
9901 gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg
9961 agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag
10021 ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
10081 tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc
10141 tcgacagacg tcgcggtgag ttcaggcttt ttcatatcgg ggtcgtcctc tccaaatgaa
10201 atgaacttcc ttatatagag gaagggtctt gcgaaggata gtgggattgt gcgtcatccc
10261 ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt
10321 ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg
10381 catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct
10441 tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc
10501 ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt
10561 tgatattctt ggagtagacg agagtgtcgt gctccaccat gttgacggat ctctaggacg
10621 cgtcctagaa gctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
10681 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
10741 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg cccgctcctt
10801 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
10861 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
10921 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
10981 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
11041 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa
11101 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
11161 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccacccc
11221 agtacattaa aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac
11281 cacaatatat cctgccacca gccagccaac agctccccga ccggcagctc ggcacaaaat
11341 caccactcga tacaggcagc ccatcagtcc gggacggcgt cagcgggaga gccgttgtaa
11401 ggcggcagac tttgctcatg ttaccgatgc tattcggaag aacggcaact aagctgccgg
11461 gtttgaaaca cggatgatct cgcggagggt agcatgttga ttgtaacgat gacagagcgt
11521 tgctgcctgt gatcaaatat catctccctc gcagagatcc gaattatcag ccttcttatt
11581 catttctcgc ttaaccgtga caggctgtcg atcttgagaa ctatgccgac ataataggaa
11641 atcgctggat aaagccgctg aggaagctga gtggcgctat ttctttagaa gtgaacgttg
11701 acgatatcaa ctcccctatc cattgctcac cgaatggtac aggtcgggga cccgaagttc
11761 cgactgtcgg cctgatgcat ccccggctga tcgaccccag atctggggct gagaaagccc
11821 agtaaggaaa caactgtagg ttcgagtcgc gagatccccc ggaaccaaag gaagtaggtt
11881 aaacccgctc cgatcaggcc gagccacgcc aggccgagaa cattggttcc tgtaggcatc
11941 gggattggcg gatcaaacac taaagctact ggaacgagca gaagtcctcc ggccgccagt
12001 tgccaggcgg taaaggtgag cagaggcacg ggaggttgcc acttgcgggt cagcacggtt
12061 ccgaacgcca tggaaaccgc ccccgccagg cccgctgcga cgccgacagg atctagcgct
12121 gcgtttggtg tcaacaccaa cagcgccacg cccgcagttc cgcaaatagc ccccaggacc
12181 gccatcaatc gtatcgggct acctagcaga gcggcagaga tgaacacgac catcagcggc
12241 tgcacagcgc ctaccgtcgc cgcgaccccg cccggcaggc ggtagaccga aataaacaac
12301 aagctccaga atagcgaaat attaagtgcg ccgaggatga agatgcgcat ccaccagatt
12361 cccgttggaa tctgtcggac gatcatcacg agcaataaac ccgccggcaa cgcccgcagc
12421 agcataccgg cgacccctcg gcctcgctgt tcgggctcca cgaaaacgcc ggacagatgc
12481 gccttgtgag cgtccttggg gccgtcctcc tgtttgaaga ccgacagccc aatgatctcg
12541 ccgtcgatgt aggcgccgaa tgccacggca tctcgcaacc gttcagcgaa cgcctccatg
12601 ggctttttct cctcgtgctc gtaaacggac ccgaacatct ctggagcttt cttcagggcc
12661 gacaatcgga tctcgcggaa atcctgcacg tcggccgctc caagccgtcg aatctgagcc
12721 ttaatcacaa ttgtcaattt taatcctctg tttatcggca gttcgtagag cgcgccgtgc
12781 gtcccgagcg atactgagcg aagcaagtgc gtcgagcagt gcccgcttgt tcctgaaatg
12841 ccagtaaagc gctggctgct gaacccccag ccggaactga ccccacaagg ccctagcgtt
12901 tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg cctcgcaact
12961 cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc
13021 acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt
13081 cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg aatttcgtgt
13141 agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg caacgggacg
13201 ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc
13261 caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg
13321 ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg
13381 tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc tgctgccaca
13441 ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc acgtccttgt
13501 tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg
13561 tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg
13621 caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg
13681 cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt tttcgcttct
13741 tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct
13801 gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca gggggagcca
13861 gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gaccatcgag ccgacggact
13921 ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg
13981 cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac gtccgattca
14041 ttcaccctcc ttgcgggatt gccccgactc acgccggggc aatgtgccct tattcctgat
14101 ttgacccgcc tggtgccttg gtgtccagat aatccacctt atcggcaatg aagtcggtcc
14161 cgtagaccgt ctggccgtcc ttctcgtact tggtattccg aatcttgccc tgcacgaata
14221 ccagcgaccc cttgcccaaa tacttgccgt gggcctcggc ctgagagcca aaacacttga
14281 tgcggaagaa gtcggtgcgc tcctgcttgt cgccggcatc gttgcgccac atctaggtac
14341 taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc
14401 agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg
14461 acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag
14521 ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag
14581 acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta
14641 aatggagtgt cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag
14701 taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg
14761 atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt
14821 tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc
14881 cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat
14941 agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc
15001 atttttaaat ataggttttc attttctccc accagcttat ataccttagc aggagacatt
15061 ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt
15121 ctcattttag ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag
15181 aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat
15241 accagaaaac agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg
15301 agccgatttt gaaaccacaa ttatgggtga tgctgccaac ttactgattt agtgtatgat
15361 ggtgtttttg aggtgctcca gtggcttctg tgtctatcag ctgtccctcc tgttcagcta
15421 ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctat ctctgctctc
15481 actgccgtaa aacatggcaa ctgcagttca cttacaccgc ttctcaaccc ggtacgcacc
15541 agaaaatcat tgatatggcc atgaatggcg ttggatgccg ggcaacagcc cgcattatgg
15601 gcgttggcct caacacgatt ttacgtcact taaaaaactc aggccgcagt cggtaacctc
15661 gcgcatacag ccgggcagtg acgtcatcgt ctgcgcggaa atggacgaac agtggggcta
15721 tgtcggggct aaatcgcgcc agcgctggct gttttacgcg tatgacagtc tccggaagac
15781 ggttgttgcg cacgtattcg gtgaacgcac tatggcgacg ctggggcgtc ttatgagcct
15841 gctgtcaccc tttgacgtgg tgatatggat gacggatggc tggccgctgt atgaatcccg
15901 cctgaaggga aagctgcacg taatcagcaa gcgatatacg cagcgaattg agcggcataa
15961 cctgaatctg aggcagcacc tggcacggct gggacggaag tcgctgtcgt tctcaaaatc
16021 ggtggagctg catgacaaag tcatcgggca ttatctgaac ataaaacact atcaataagt
16081 tggagtcatt acccaattat gatagaattt acaagctata aggttattgt cctgggtttc
16141 aagcattagt ccatgcaagt ttttatgctt tgcccattct atagatatat tgataagcgc
16201 gctgcctatg ccttgccccc tgaaatcctt acatacggcg atatcttcta tataaaagat
16261 atattatctt atcagtattg tcaatatatt caaggcaatc tgcctcctca tcctcttcat
16321 cctcttcgtc ttggtagctt tttaaatatg gcgcttcata gagtaattct gtaaaggtcc
16381 aattctcgtt ttcatacctc ggtataatct tacctatcac ctcaaatggt tcgctgggtt
16441 tatcgcaccc ccgaacacga gcacggcacc cgcgaccact atgccaagaa tgcccaaggt
16501 aaaaattgcc ggccccgcca tgaagtccgt gaatgccccg acggccgaag tgaagggcag
16561 gccgccaccc aggccgccgc cctcactgcc cggcacctgg tcgctgaatg tcgatgccag
16621 cacctgcggc acgtcaatgc ttccgggcgt cgcgctcggg ctgatcgccc atcccgttac
16681 tgccccgatc ccggcaatgg caaggactgc cagcgctgcc atttttgggg tgaggccgtt
16741 cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg
16801 ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct
16861 ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt
16921 ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct
16981 caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc
17041 gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc
17101 ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt
17161 gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg
17221 ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg
17281 ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca cacggcttcg
17341 acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa
17401 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
| P2857/pENTR223-RBBP4（2同义突变）人源基因质粒 |
| P2858/pENTR223-FKBP8（179-1239bp）人源基因质粒 |
| P2859/pENTR223-FAM172A人源基因质粒 |
| P2860/pENTR223-TRNT1（88-1305bp）人源基因质粒 |
| P2861/pENTR223-SERPINF1（2同义突变）人源基因质粒 |
| P2862/pENTR223-PSMC4人源基因质粒 |
| P2863/pENTR223-NKAP(2点突变）人源基因质粒 |
| P2864/pENTR223-IKBKG人源基因质粒 |
| P2865/pENTR223-CCDC91-G850A人源基因质粒 |
| P2866/pENTR223-PELI2人源基因质粒 |
| P2867/pENTR223-PHKG2人源基因质粒 |
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pGWB18植物表达质粒
￥100 - 1000
