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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：
pGWB4
- 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：pGWB4植物表达质粒
别称: pGWB4
质粒属性
质粒简介
GWB4是一个植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17102 bp ds-DNA circular SYN 25-JAN-2018
FEATURES Location/Qualifiers
source 1..17102
/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=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"
protein_bind 4977..5101
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 5138..5168
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 5222..5881
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 6223..6528
/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(6569..6693)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 6698..7417
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITHGMDELYK"
terminator 7442..7694
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8123..8375
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(8843..9868)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMD-AIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(9878..10222)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 10966..10990
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12586..13734)
/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(14033..14827)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15019..15786
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16409..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 cctgcaggct ctaatcacaa
4981 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
5041 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
5101 ggcggcccct cgaggggccg cattaggcac cccaggcttt acactttatg cttccggctc
5161 gtataatgtg tggattttga gttaggatcc ggcgagattt tcaggagcta aggaagctaa
5221 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
5281 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
5341 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
5401 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
5461 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
5521 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
5581 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
5641 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
5701 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
5761 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca
5821 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
5881 aacgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
5941 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtgtgct
6001 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
6061 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
6121 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
6181 aaatgaacgg ctcttttgct gacgagaaca gggactggtg aaatgcagtt taaggtttac
6241 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
6301 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
6361 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
6421 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
6481 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
6541 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
6601 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
6661 cgtttctcgt tcagctttct tgtacaaagt ggtgatcatg gtgagcaagg gcgaggagct
6721 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtgaacg gccacaagtt
6781 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
6841 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg
6901 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
6961 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
7021 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
7081 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
7141 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
7201 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
7261 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
7321 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
7381 cgggatcact cacggcatgg acgagctgta caagtaaagc ggcccgagct cgaatttccc
7441 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
7501 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
7561 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
7621 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
7681 tatgttacta gatcgggaat tagcttcatc aacgcaagac atgcgcacga ccgtctgaca
7741 ggagaggaat ttccgacgag cacagaaagg acttgctctt ggacgtaggc ctatttctca
7801 ggcacatgta tcaagtgttc ggacgtgggt tttcgatggt gtatcagccg ccgccaactg
7861 ggagatgagg aggctttctt ggggggcagt cagcagttca tttcacaaga cagaggaact
7921 tgtaaggaga tgcactgatt tatcttggcg caaaccagca ggacgaatta gtgggaatag
7981 cccgcgaata tctaagttat gcctgtcggc atgagcagaa acttccaatt cgaaacagtt
8041 tggagaggtt gtttttgggc ataccttttg ttagtcagcc tctcgattgc tcatcgtcat
8101 tacacagtac cgaagtttga tcgatctagt aacatagatg acaccgcgcg cgataattta
8161 tcctagtttg cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact
8221 ctaatcataa aaacccatct cataaataac gtcatgcatt acatgttaat tattacatgc
8281 ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattcaat
8341 cttaagaaac tttattgcca aatgtttgaa cgatctgctt cgacgcactc cttctttact
8401 ccaccatctc gtccttattg aaaacgtggg tagcaccaaa acgaatcaag tcgctggaac
8461 tgaagttacc aatcacgctg gatgatttgc cagttggatt aatcttgcct ttccccgcat
8521 gaataatatt gatgaatgca tgcgtgaggg gtatttcgat tttggcaata gctgcaattg
8581 ccgcgacatc ctccaacgag cataattctt cagaaaaata gcgatgttcc atgttgtcag
8641 ggcatgcatg atgcacgtta tgaggtgacg gtgctaggca gtattccctc aaagtttcat
8701 agtcagtatc atattcatca ttgcattcct gcaagagaga attgagacgc aatccacacg
8761 ctgcggcaac cttccggcgt tcgtggtcta tttgctcttg gacgttgcaa acgtaagtgt
8821 tggatcccgg tcggcatcta ctctattcct ttgccctcgg acgagtgctg gggcgtcggt
8881 ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg
8941 cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct
9001 gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac
9061 caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct
9121 cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg
9181 cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc
9241 ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt
9301 cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga
9361 cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga
9421 aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg
9481 tctggctaag atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag
9541 cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc
9601 aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg
9661 ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta
9721 cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct
9781 ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag
9841 acgtcgcggt gagttcaggc tttttcatat cggggtcgtc ctctccaaat gaaatgaact
9901 tccttatata gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc
9961 agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc
10021 cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg
10081 aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac
10141 tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat
10201 taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt
10261 cttggagtag acgagagtgt cgtgctccac catgttgacg gatctctagg acgcgtccta
10321 gaagctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
10381 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
10441 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgcccgctc ctttcgcttt
10501 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
10561 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
10621 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
10681 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
10741 gggctattct tttgatttat aagggatttt gccgatttcg gaaccaccat caaacaggat
10801 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
10861 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cccagtacat
10921 taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata
10981 tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact
11041 cgatacaggc agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca
11101 gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa
11161 acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc
11221 tgtgatcaaa tatcatctcc ctcgcagaga tccgaattat cagccttctt attcatttct
11281 cgcttaaccg tgacaggctg tcgatcttga gaactatgcc gacataatag gaaatcgctg
11341 gataaagccg ctgaggaagc tgagtggcgc tatttcttta gaagtgaacg ttgacgatat
11401 caactcccct atccattgct caccgaatgg tacaggtcgg ggacccgaag ttccgactgt
11461 cggcctgatg catccccggc tgatcgaccc cagatctggg gctgagaaag cccagtaagg
11521 aaacaactgt aggttcgagt cgcgagatcc cccggaacca aaggaagtag gttaaacccg
11581 ctccgatcag gccgagccac gccaggccga gaacattggt tcctgtaggc atcgggattg
11641 gcggatcaaa cactaaagct actggaacga gcagaagtcc tccggccgcc agttgccagg
11701 cggtaaaggt gagcagaggc acgggaggtt gccacttgcg ggtcagcacg gttccgaacg
11761 ccatggaaac cgcccccgcc aggcccgctg cgacgccgac aggatctagc gctgcgtttg
11821 gtgtcaacac caacagcgcc acgcccgcag ttccgcaaat agcccccagg accgccatca
11881 atcgtatcgg gctacctagc agagcggcag agatgaacac gaccatcagc ggctgcacag
11941 cgcctaccgt cgccgcgacc ccgcccggca ggcggtagac cgaaataaac aacaagctcc
12001 agaatagcga aatattaagt gcgccgagga tgaagatgcg catccaccag attcccgttg
12061 gaatctgtcg gacgatcatc acgagcaata aacccgccgg caacgcccgc agcagcatac
12121 cggcgacccc tcggcctcgc tgttcgggct ccacgaaaac gccggacaga tgcgccttgt
12181 gagcgtcctt ggggccgtcc tcctgtttga agaccgacag cccaatgatc tcgccgtcga
12241 tgtaggcgcc gaatgccacg gcatctcgca accgttcagc gaacgcctcc atgggctttt
12301 tctcctcgtg ctcgtaaacg gacccgaaca tctctggagc tttcttcagg gccgacaatc
12361 ggatctcgcg gaaatcctgc acgtcggccg ctccaagccg tcgaatctga gccttaatca
12421 caattgtcaa ttttaatcct ctgtttatcg gcagttcgta gagcgcgccg tgcgtcccga
12481 gcgatactga gcgaagcaag tgcgtcgagc agtgcccgct tgttcctgaa atgccagtaa
12541 agcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg
12601 caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca
12661 ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag
12721 gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat
12781 ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc
12841 gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt
12901 gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg
12961 gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt
13021 gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt
13081 gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc
13141 gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg
13201 gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag
13261 ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc
13321 gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg
13381 cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt
13441 catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcgag
13501 acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac
13561 gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt
13621 ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa
13681 ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc
13741 tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc
13801 gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac
13861 cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga
13921 ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa
13981 gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg tactaaaaca
14041 attcatccag taaaatataa tattttattt tctcccaatc aggcttgatc cccagtaagt
14101 caaaaaatag ctcgacatac tgttcttccc cgatatcctc cctgatcgac cggacgcaga
14161 aggcaatgtc ataccacttg tccgccctgc cgcttctccc aagatcaata aagccactta
14221 ctttgccatc tttcacaaag atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt
14281 cctcttcggg cttttccgtc tttaaaaaat catacagctc gcgcggatct ttaaatggag
14341 tgtcttcttc ccagttttcg caatccacat cggccagatc gttattcagt aagtaatcca
14401 attcggctaa gcggctgtct aagctattcg tatagggaca atccgatatg tcgatggagt
14461 gaaagagcct gatgcactcc gcatacagct cgataatctt ttcagggctt tgttcatctt
14521 catactcttc cgagcaaagg acgccatcgg cctcactcat gagcagattg ctccagccat
14581 catgccgttc aaagtgcagg acctttggaa caggcagctt tccttccagc catagcatca
14641 tgtccttttc ccgttccaca tcataggtgg tccctttata ccggctgtcc gtcattttta
14701 aatataggtt ttcattttct cccaccagct tatatacctt agcaggagac attccttccg
14761 tatcttttac gcagcggtat ttttcgatca gttttttcaa ttccggtgat attctcattt
14821 tagccattta ttatttcctt cctcttttct acagtattta aagatacccc aagaagctaa
14881 ttataacaag acgaactcca attcactgtt ccttgcattc taaaacctta aataccagaa
14941 aacagctttt tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggagccgat
15001 tttgaaacca caattatggg tgatgctgcc aacttactga tttagtgtat gatggtgttt
15061 ttgaggtgct ccagtggctt ctgtgtctat cagctgtccc tcctgttcag ctactgacgg
15121 ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tatctctgct ctcactgccg
15181 taaaacatgg caactgcagt tcacttacac cgcttctcaa cccggtacgc accagaaaat
15241 cattgatatg gccatgaatg gcgttggatg ccgggcaaca gcccgcatta tgggcgttgg
15301 cctcaacacg attttacgtc acttaaaaaa ctcaggccgc agtcggtaac ctcgcgcata
15361 cagccgggca gtgacgtcat cgtctgcgcg gaaatggacg aacagtgggg ctatgtcggg
15421 gctaaatcgc gccagcgctg gctgttttac gcgtatgaca gtctccggaa gacggttgtt
15481 gcgcacgtat tcggtgaacg cactatggcg acgctggggc gtcttatgag cctgctgtca
15541 ccctttgacg tggtgatatg gatgacggat ggctggccgc tgtatgaatc ccgcctgaag
15601 ggaaagctgc acgtaatcag caagcgatat acgcagcgaa ttgagcggca taacctgaat
15661 ctgaggcagc acctggcacg gctgggacgg aagtcgctgt cgttctcaaa atcggtggag
15721 ctgcatgaca aagtcatcgg gcattatctg aacataaaac actatcaata agttggagtc
15781 attacccaat tatgatagaa tttacaagct ataaggttat tgtcctgggt ttcaagcatt
15841 agtccatgca agtttttatg ctttgcccat tctatagata tattgataag cgcgctgcct
15901 atgccttgcc ccctgaaatc cttacatacg gcgatatctt ctatataaaa gatatattat
15961 cttatcagta ttgtcaatat attcaaggca atctgcctcc tcatcctctt catcctcttc
16021 gtcttggtag ctttttaaat atggcgcttc atagagtaat tctgtaaagg tccaattctc
16081 gttttcatac ctcggtataa tcttacctat cacctcaaat ggttcgctgg gtttatcgca
16141 cccccgaaca cgagcacggc acccgcgacc actatgccaa gaatgcccaa ggtaaaaatt
16201 gccggccccg ccatgaagtc cgtgaatgcc ccgacggccg aagtgaaggg caggccgcca
16261 cccaggccgc cgccctcact gcccggcacc tggtcgctga atgtcgatgc cagcacctgc
16321 ggcacgtcaa tgcttccggg cgtcgcgctc gggctgatcg cccatcccgt tactgccccg
16381 atcccggcaa tggcaaggac tgccagcgct gccatttttg gggtgaggcc gttcgcggcc
16441 gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga
16501 agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa
16561 aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa
16621 aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt
16681 caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc
16741 tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct
16801 tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc
16861 tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc
16921 gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt
16981 ttccgcgagg tatccacaac gccggcggcc gcggtgtctc gcacacggct tcgacggcgt
17041 ttctggcgcg tttgcagggc catagacggc cgccagccca gcggcgaggg caaccagccc
17101 gg
//
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：pGWB4植物表达质粒
别称: pGWB4
| 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： | Promoter,GateWay |
| 原核抗性： | Kan |
| 筛选标记： | Neo/G418,Hyg |
| 荧光蛋白： | 绿色 |
质粒简介
GWB4是一个植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17102 bp ds-DNA circular SYN 25-JAN-2018
FEATURES Location/Qualifiers
source 1..17102
/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=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"
protein_bind 4977..5101
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 5138..5168
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 5222..5881
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 6223..6528
/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(6569..6693)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 6698..7417
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITHGMDELYK"
terminator 7442..7694
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8123..8375
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(8843..9868)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMD-AIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(9878..10222)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 10966..10990
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12586..13734)
/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(14033..14827)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15019..15786
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16409..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 cctgcaggct ctaatcacaa
4981 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
5041 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
5101 ggcggcccct cgaggggccg cattaggcac cccaggcttt acactttatg cttccggctc
5161 gtataatgtg tggattttga gttaggatcc ggcgagattt tcaggagcta aggaagctaa
5221 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
5281 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
5341 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
5401 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
5461 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
5521 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
5581 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
5641 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
5701 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
5761 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca
5821 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
5881 aacgcgtgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
5941 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtgtgct
6001 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
6061 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
6121 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
6181 aaatgaacgg ctcttttgct gacgagaaca gggactggtg aaatgcagtt taaggtttac
6241 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
6301 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
6361 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
6421 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
6481 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
6541 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
6601 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
6661 cgtttctcgt tcagctttct tgtacaaagt ggtgatcatg gtgagcaagg gcgaggagct
6721 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtgaacg gccacaagtt
6781 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
6841 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg
6901 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
6961 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
7021 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
7081 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
7141 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
7201 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
7261 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
7321 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
7381 cgggatcact cacggcatgg acgagctgta caagtaaagc ggcccgagct cgaatttccc
7441 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
7501 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
7561 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
7621 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
7681 tatgttacta gatcgggaat tagcttcatc aacgcaagac atgcgcacga ccgtctgaca
7741 ggagaggaat ttccgacgag cacagaaagg acttgctctt ggacgtaggc ctatttctca
7801 ggcacatgta tcaagtgttc ggacgtgggt tttcgatggt gtatcagccg ccgccaactg
7861 ggagatgagg aggctttctt ggggggcagt cagcagttca tttcacaaga cagaggaact
7921 tgtaaggaga tgcactgatt tatcttggcg caaaccagca ggacgaatta gtgggaatag
7981 cccgcgaata tctaagttat gcctgtcggc atgagcagaa acttccaatt cgaaacagtt
8041 tggagaggtt gtttttgggc ataccttttg ttagtcagcc tctcgattgc tcatcgtcat
8101 tacacagtac cgaagtttga tcgatctagt aacatagatg acaccgcgcg cgataattta
8161 tcctagtttg cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact
8221 ctaatcataa aaacccatct cataaataac gtcatgcatt acatgttaat tattacatgc
8281 ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattcaat
8341 cttaagaaac tttattgcca aatgtttgaa cgatctgctt cgacgcactc cttctttact
8401 ccaccatctc gtccttattg aaaacgtggg tagcaccaaa acgaatcaag tcgctggaac
8461 tgaagttacc aatcacgctg gatgatttgc cagttggatt aatcttgcct ttccccgcat
8521 gaataatatt gatgaatgca tgcgtgaggg gtatttcgat tttggcaata gctgcaattg
8581 ccgcgacatc ctccaacgag cataattctt cagaaaaata gcgatgttcc atgttgtcag
8641 ggcatgcatg atgcacgtta tgaggtgacg gtgctaggca gtattccctc aaagtttcat
8701 agtcagtatc atattcatca ttgcattcct gcaagagaga attgagacgc aatccacacg
8761 ctgcggcaac cttccggcgt tcgtggtcta tttgctcttg gacgttgcaa acgtaagtgt
8821 tggatcccgg tcggcatcta ctctattcct ttgccctcgg acgagtgctg gggcgtcggt
8881 ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg
8941 cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct
9001 gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac
9061 caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct
9121 cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg
9181 cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc
9241 ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt
9301 cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga
9361 cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga
9421 aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg
9481 tctggctaag atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag
9541 cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc
9601 aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg
9661 ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta
9721 cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct
9781 ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag
9841 acgtcgcggt gagttcaggc tttttcatat cggggtcgtc ctctccaaat gaaatgaact
9901 tccttatata gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc
9961 agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc
10021 cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg
10081 aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac
10141 tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat
10201 taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt
10261 cttggagtag acgagagtgt cgtgctccac catgttgacg gatctctagg acgcgtccta
10321 gaagctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
10381 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
10441 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgcccgctc ctttcgcttt
10501 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
10561 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
10621 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
10681 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
10741 gggctattct tttgatttat aagggatttt gccgatttcg gaaccaccat caaacaggat
10801 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
10861 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cccagtacat
10921 taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata
10981 tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact
11041 cgatacaggc agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca
11101 gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa
11161 acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc
11221 tgtgatcaaa tatcatctcc ctcgcagaga tccgaattat cagccttctt attcatttct
11281 cgcttaaccg tgacaggctg tcgatcttga gaactatgcc gacataatag gaaatcgctg
11341 gataaagccg ctgaggaagc tgagtggcgc tatttcttta gaagtgaacg ttgacgatat
11401 caactcccct atccattgct caccgaatgg tacaggtcgg ggacccgaag ttccgactgt
11461 cggcctgatg catccccggc tgatcgaccc cagatctggg gctgagaaag cccagtaagg
11521 aaacaactgt aggttcgagt cgcgagatcc cccggaacca aaggaagtag gttaaacccg
11581 ctccgatcag gccgagccac gccaggccga gaacattggt tcctgtaggc atcgggattg
11641 gcggatcaaa cactaaagct actggaacga gcagaagtcc tccggccgcc agttgccagg
11701 cggtaaaggt gagcagaggc acgggaggtt gccacttgcg ggtcagcacg gttccgaacg
11761 ccatggaaac cgcccccgcc aggcccgctg cgacgccgac aggatctagc gctgcgtttg
11821 gtgtcaacac caacagcgcc acgcccgcag ttccgcaaat agcccccagg accgccatca
11881 atcgtatcgg gctacctagc agagcggcag agatgaacac gaccatcagc ggctgcacag
11941 cgcctaccgt cgccgcgacc ccgcccggca ggcggtagac cgaaataaac aacaagctcc
12001 agaatagcga aatattaagt gcgccgagga tgaagatgcg catccaccag attcccgttg
12061 gaatctgtcg gacgatcatc acgagcaata aacccgccgg caacgcccgc agcagcatac
12121 cggcgacccc tcggcctcgc tgttcgggct ccacgaaaac gccggacaga tgcgccttgt
12181 gagcgtcctt ggggccgtcc tcctgtttga agaccgacag cccaatgatc tcgccgtcga
12241 tgtaggcgcc gaatgccacg gcatctcgca accgttcagc gaacgcctcc atgggctttt
12301 tctcctcgtg ctcgtaaacg gacccgaaca tctctggagc tttcttcagg gccgacaatc
12361 ggatctcgcg gaaatcctgc acgtcggccg ctccaagccg tcgaatctga gccttaatca
12421 caattgtcaa ttttaatcct ctgtttatcg gcagttcgta gagcgcgccg tgcgtcccga
12481 gcgatactga gcgaagcaag tgcgtcgagc agtgcccgct tgttcctgaa atgccagtaa
12541 agcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg
12601 caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca
12661 ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag
12721 gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat
12781 ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc
12841 gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt
12901 gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg
12961 gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt
13021 gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt
13081 gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc
13141 gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg
13201 gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag
13261 ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc
13321 gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg
13381 cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt
13441 catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcgag
13501 acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac
13561 gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt
13621 ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa
13681 ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc
13741 tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc
13801 gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac
13861 cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga
13921 ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa
13981 gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg tactaaaaca
14041 attcatccag taaaatataa tattttattt tctcccaatc aggcttgatc cccagtaagt
14101 caaaaaatag ctcgacatac tgttcttccc cgatatcctc cctgatcgac cggacgcaga
14161 aggcaatgtc ataccacttg tccgccctgc cgcttctccc aagatcaata aagccactta
14221 ctttgccatc tttcacaaag atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt
14281 cctcttcggg cttttccgtc tttaaaaaat catacagctc gcgcggatct ttaaatggag
14341 tgtcttcttc ccagttttcg caatccacat cggccagatc gttattcagt aagtaatcca
14401 attcggctaa gcggctgtct aagctattcg tatagggaca atccgatatg tcgatggagt
14461 gaaagagcct gatgcactcc gcatacagct cgataatctt ttcagggctt tgttcatctt
14521 catactcttc cgagcaaagg acgccatcgg cctcactcat gagcagattg ctccagccat
14581 catgccgttc aaagtgcagg acctttggaa caggcagctt tccttccagc catagcatca
14641 tgtccttttc ccgttccaca tcataggtgg tccctttata ccggctgtcc gtcattttta
14701 aatataggtt ttcattttct cccaccagct tatatacctt agcaggagac attccttccg
14761 tatcttttac gcagcggtat ttttcgatca gttttttcaa ttccggtgat attctcattt
14821 tagccattta ttatttcctt cctcttttct acagtattta aagatacccc aagaagctaa
14881 ttataacaag acgaactcca attcactgtt ccttgcattc taaaacctta aataccagaa
14941 aacagctttt tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggagccgat
15001 tttgaaacca caattatggg tgatgctgcc aacttactga tttagtgtat gatggtgttt
15061 ttgaggtgct ccagtggctt ctgtgtctat cagctgtccc tcctgttcag ctactgacgg
15121 ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tatctctgct ctcactgccg
15181 taaaacatgg caactgcagt tcacttacac cgcttctcaa cccggtacgc accagaaaat
15241 cattgatatg gccatgaatg gcgttggatg ccgggcaaca gcccgcatta tgggcgttgg
15301 cctcaacacg attttacgtc acttaaaaaa ctcaggccgc agtcggtaac ctcgcgcata
15361 cagccgggca gtgacgtcat cgtctgcgcg gaaatggacg aacagtgggg ctatgtcggg
15421 gctaaatcgc gccagcgctg gctgttttac gcgtatgaca gtctccggaa gacggttgtt
15481 gcgcacgtat tcggtgaacg cactatggcg acgctggggc gtcttatgag cctgctgtca
15541 ccctttgacg tggtgatatg gatgacggat ggctggccgc tgtatgaatc ccgcctgaag
15601 ggaaagctgc acgtaatcag caagcgatat acgcagcgaa ttgagcggca taacctgaat
15661 ctgaggcagc acctggcacg gctgggacgg aagtcgctgt cgttctcaaa atcggtggag
15721 ctgcatgaca aagtcatcgg gcattatctg aacataaaac actatcaata agttggagtc
15781 attacccaat tatgatagaa tttacaagct ataaggttat tgtcctgggt ttcaagcatt
15841 agtccatgca agtttttatg ctttgcccat tctatagata tattgataag cgcgctgcct
15901 atgccttgcc ccctgaaatc cttacatacg gcgatatctt ctatataaaa gatatattat
15961 cttatcagta ttgtcaatat attcaaggca atctgcctcc tcatcctctt catcctcttc
16021 gtcttggtag ctttttaaat atggcgcttc atagagtaat tctgtaaagg tccaattctc
16081 gttttcatac ctcggtataa tcttacctat cacctcaaat ggttcgctgg gtttatcgca
16141 cccccgaaca cgagcacggc acccgcgacc actatgccaa gaatgcccaa ggtaaaaatt
16201 gccggccccg ccatgaagtc cgtgaatgcc ccgacggccg aagtgaaggg caggccgcca
16261 cccaggccgc cgccctcact gcccggcacc tggtcgctga atgtcgatgc cagcacctgc
16321 ggcacgtcaa tgcttccggg cgtcgcgctc gggctgatcg cccatcccgt tactgccccg
16381 atcccggcaa tggcaaggac tgccagcgct gccatttttg gggtgaggcc gttcgcggcc
16441 gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga
16501 agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa
16561 aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa
16621 aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt
16681 caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc
16741 tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct
16801 tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc
16861 tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc
16921 gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt
16981 ttccgcgagg tatccacaac gccggcggcc gcggtgtctc gcacacggct tcgacggcgt
17041 ttctggcgcg tttgcagggc catagacggc cgccagccca gcggcgaggg caaccagccc
17101 gg
//
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
| P2781/pENTR223-THUMPD1人源基因质粒 |
| P2782/pENTR223-AFF4（1-1087bp）人源基因质粒 |
| P2783/pENTR223-RPF1-A25G人源基因质粒 |
| P2784/pENTR223-ARHGAP29（1-1145bp）人源基因质粒 |
| P2785/pENTR223-PCYT2人源基因质粒 |
| P2786/pENTR223-PPID（倒数第20碱基A缺失）人源基因质粒 |
| P2787/pENTR223-SURF6人源基因质粒 |
| P2788/pENTR223-PSMD4人源基因质粒 |
| P2789/pENTR223-DAZAP2人源基因质粒 |
| P2790/pENTR223-MUL1（3点突变）人源基因质粒 |
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pGWB4植物表达质粒
￥100 - 1000
