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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：
pK7GWIWG2II-RedRoot
- 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：pK7GWIWG2II-RedRoot植物表达质粒
别称: pK7GWIWG2II-RedRoot
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 15413 bp ds-DNA circular SYN 20-JUL-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pK7GWIWG2 II-RedRoot
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15413)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 19, 2016 from SnapGene Viewer 3.2.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..15413
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 65..317
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 2772..2896
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR1"
/note="recombination site for the Gateway(R) LR reaction"
promoter 2921..2951
/note="lac UV5 promoter"
/note="E. coli lac promoter with an ""up"" mutation"
CDS 3303..3608
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="ccdB"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(3649..3773)
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR2"
/note="recombination site for the Gateway(R) LR reaction"
CDS 4148..4807
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/note="CmR"
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
protein_bind 5210..5334
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR2"
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(5375..5680)
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="ccdB"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
promoter complement(6032..6062)
/note="lac UV5 promoter"
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind complement(6087..6211)
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR1"
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(6320..6664)
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 7357..7381
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 8681..9310
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 9744..10811
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 RepA"
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 10877..11071
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 11415..11555
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(11741..12329)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12573..13364)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/note="SmR"
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_feature 13892..13916
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
terminator 14009..14261
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(14301..15095)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(15129..15308)
/note="NOS promoter"
/note="nopaline synthase promoter"
ORIGIN
1 ctagatgcat gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttgggc
61 ccccgatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat
121 attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat
181 ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga
241 aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc
301 caaatgtttg aacgatcggg aattggatcc tacaggaaca ggtggtggcg gccctcggtg
361 cgctcgtact gctccacgat ggtgtagtcc tcgtttgtgg gaggtgatgt ccagcttgga
421 gtccacgtag tagtagccgg gcagctgcac gggcttcttg gccatgtaga tggacttgaa
481 ctccaccagg tagtggccgc cgtccttcag cttcagggcc ttgtggatct cgcccttcag
541 cacgccgtcg cgggggtaca ggcgctcggt ggaggcctcc cagcccatgg tcttcttctg
601 cattacgggg ccgtcggagg ggaagttcac gccgatgaac ttcaccttgt agatgaagca
661 gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat
721 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc ttgtagtcgg ggatgtcggc
781 ggggtgcttc acgtacacct tggagccgta ctggaactgg ggggacagga tgtcccaggc
841 gaagggcagg gggccgccct tggtcacctt cagcttcacg gtgttgtggc cctcgtaggg
901 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggtgc cctccatgcg
961 caccttgaag cgcatgaact ccttgatgac gttcttggag gagcgcgcca tggcaaagat
1021 ctgcatctgt taatcagaaa aactcagatt aatcgacaaa ttcgatcgca caaactagaa
1081 actaacacca gatctagata gaaatcacaa atcgaagagt aattattcga caaaactcaa
1141 attatttgaa caaatcggat gatatctatg aaaccctaat cgagaattaa gatgatatct
1201 aacgatcaaa cccagaaaat cgtcttcgat ctaagattaa cagaatctaa accaaagaac
1261 atatacgaaa ttgggatcga acgaaaacaa aatcgaagat tttgagagaa taaggaacac
1321 agaaatttac cttgatcacg gtagagagaa ttgagagaaa gtttttaaga ttttgagaaa
1381 ttgaaatctg aattgtgaag aagaagagct ctttgggtat tgttttatag aagaagaaga
1441 agaaaagacg aggacgacta ggtcacgaga aagctaaggc ggtgaagcaa tagctaataa
1501 taaaatgaca cgtgtattga gcgttgttta cacgcaaagt tgtttttggc taattgcctt
1561 atttttaggt tgaggaaaag tatttgtgct ttgagttgat aaacacgact cgtgtgtgcc
1621 ggctgcaacc actttgacgc cgtttattac tggacttcgt ccgacaacca caatttcctt
1681 aacggtcgtc ataagatcca gccgttgaga tttaacgatc gttacgattt atattttttt
1741 agcattatcg ttttattttt taaatatacg gtggagctga aaattggcaa taattgaacc
1801 gtgggtccca ctgcattgaa gcgtatttcg tattttctag aattcttcgt gctttatttc
1861 ttttcctttt tgtttttttt tgccatttat ctaatgcaag tgggcttata aaatcagtga
1921 atttcttgga aaagtaactt ctttatcgta taacatattg tgaaattatc catttctttt
1981 aattttttta gtgttattgg atatttttgt atgattattg atttgcatag gataatgact
2041 tttgtatcaa gttggtgaac aagtctcgtt aaaaaaggca agtggtttgg tgactcgatt
2101 tattcttgtt atttaattca tatatcaatg gatcttattt ggggcctggt ccatatttaa
2161 cactcgtgtt cagtccaatg accaataata ttttttcatt aataacaatg taacaagaat
2221 gatacacaaa acattctttg aataagttcg ctatgaagaa gggaacttat ccggtcctag
2281 atcatcagtt catacaaacc tccatagagt tcaacatctt aaacaagaat atcctgatct
2341 gaagaatgtg gaggctttag tcccttggat acttgggagg ctgtggaaga acagaaacga
2401 gctggtgctt aaagggaggg aatttggaac caatgaggta ttagtaagga cacaagaaga
2461 tgcagatgag tggattagaa ggaaagaggc tcagaatgta aggaaagcac caaccacaac
2521 gacgtcgcat gcctgcaggt cactggattt tggttttagg aattagaaat tttattgata
2581 gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg
2641 aaaccctata agaaccctaa ttcccttatc tgggaactac tcacacatta ttctggagaa
2701 aaatagagag agatagattt gtagagagag actggtgatt tttgcggact ctagcatggc
2761 cgcgggatat cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata
2821 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
2881 atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg
2941 ctcgtataat gtgtggattt tgagttagga tccggcttac taaaagccag ataacagtat
3001 gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt
3061 atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt gacagttgac agcgacagct
3121 atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca
3181 gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc
3241 tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca gggactggtg
3301 aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
3361 tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac
3421 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
3481 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
3541 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
3601 gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacca tagtgactgg
3661 atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata
3721 ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtgatatca
3781 ctagtgcggc cgcctgcagg tcgaccatat ggtcgacctg caggcggccg cactagtgat
3841 gctgttatgt tcagtgtcaa gctgacctgc aaacacgtta aatgctaaga agttagaata
3901 tatgagacac gttaactggt atatgaataa gctgtaaata accgagtata aactcattaa
3961 ctaatatcac ctctagagta taatataatc aaattcgaca atttgacttt caagagtagg
4021 ctaatgtaaa atctttatat atttctacaa tgttcaaaga aacagttgca tctaaacccc
4081 tatggccatc aaattcaatg aacgctaagc tgatccggcg agattttcag gagctaagga
4141 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
4201 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
4261 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
4321 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
4381 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
4441 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
4501 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
4561 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
4621 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
4681 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tctgtgatgg
4741 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
4801 ggcgtaaacg cgtggatcag cttaatatga ctctcaataa agtctcatac caacaagtgc
4861 caccttattc aaccatcaag aaaaaagcca aaatttatgc tactctaagg aaaacttcac
4921 taaagaagac gatttagagt gttttaccaa gaatttctgt catcttacta aacaactaaa
4981 gatcggtgtg atacaaaacc taatctcatt aaagtttatg ctaaaataag cataatttta
5041 cccactaagc gtgaccagat aaacataact cagcacacca gagcatatat attggtggct
5101 caaatcatag aaacttacag tgaagacaca gaaagccgta agaagaggca agagtatgaa
5161 accttacctc atcatttcca tgaggttgct tctgatcccg cgggatatca ccactttgta
5221 caagaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
5281 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggtcgac
5341 ctgcagactg gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa
5401 tggcgttttt gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg
5461 agaccggcac actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca
5521 ccaccgggta aagttcacgg gagactttat ctgacagcag acgtgcactg gccaggggga
5581 tcaccatccg tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat
5641 aacggctctc tcttttatag gtgtaaacct taaactgcat ttcaccagtc cctgttctcg
5701 tcagcaaaag agccgttcat ttcaataaac cgggcgacct cagccatccc ttcctgattt
5761 tccgctttcc agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc
5821 agaccggaga tattgacatc atatatgcct tgagcaactg atagctgtcg ctgtcaactg
5881 tcactgtaat acgctgcttc atagcacacc tctttttgac atacttcggg tatacatatc
5941 agtatatatt cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt
6001 tagtaagccg gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta
6061 aagcctgggg tgcctaatgc ggccgccata gtgactggat atgttgtgtt ttacagtatt
6121 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
6181 tttctcgttc agcttttttg tacaaacttg tgatatcact agtgcggccg cctgcaggtc
6241 gactagaata gtaaattgta atgttgtttg ttgtttgttt tgttgtggta attgttgtaa
6301 aaatacggat cgtcctgcag tcctctccaa atgaaatgaa cttccttata tagaggaagg
6361 gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg tcagtggaga tatcacatca
6421 atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg
6481 tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt
6541 atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg
6601 acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt
6661 ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt
6721 gtcgtgctcc accatgttga cgaagatttt cttcttgtca ttgagtcgta aaagactctg
6781 tatgaactgt tcgccagtct tcacggcgag ttctgttaga tcctcgatct gaatttttga
6841 ctccatggcc tttgattcag taggaactac tttcttagag actccaatct ctattacttg
6901 ccttggttta tgaagcaagc cttgaatcgt ccatactgga atagtacttc tgatcttgag
6961 aaatatatct ttctctgtgt tcttgatgca gttagtcctg aatcttttga ctgcatcttt
7021 aaccttcttg ggaaggtatt tgatctcctg gagattatta ctcgggtaga tcgtcttgat
7081 gagacctgcc gcgtaggcct ctctaaccat ctgtgggtca gcattctttc tgaaattgaa
7141 gaggctaatc ttctcattat cggtggtgaa catggtatcg tcaccttctc cgtcgaactt
7201 tcttcctaga tcgtagagat agagaaagtc gtccatggtg atctccgggg caaaggagat
7261 cagcttggct ctagtcgacc atatgggaga gctcaagctt agcttgagct tggatcagat
7321 tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa
7381 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta
7441 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact
7501 ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct
7561 tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt
7621 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac
7681 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc
7741 agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc
7801 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg
7861 cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc
7921 agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt
7981 agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg
8041 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc
8101 gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag
8161 atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca
8221 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg
8281 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc
8341 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc
8401 aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg
8461 tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt
8521 ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc
8581 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata
8641 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact
8701 taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca
8761 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg
8821 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga
8881 ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc
8941 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc
9001 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga
9061 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg
9121 catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg
9181 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc
9241 agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa
9301 actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc
9361 ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca
9421 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac
9481 gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca
9541 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat
9601 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg
9661 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc
9721 caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg
9781 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca
9841 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag
9901 aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg
9961 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca
10021 tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc
10081 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg
10141 tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat
10201 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac
10261 tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca
10321 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc
10381 tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa
10441 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag
10501 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca
10561 tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt
10621 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca
10681 ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg
10741 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg
10801 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc
10861 gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga
10921 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag
10981 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta
11041 aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc
11101 tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc
11161 ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc
11221 gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc
11281 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
11341 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
11401 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
11461 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
11521 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
11581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
11641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
11701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
11761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
11821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
11881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
11941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
12001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
12061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
12121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
12181 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
12241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
12301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
12361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcatgat atatctccca
12421 atttgtgtag ggcttattat gcacgcttaa aaataataaa agcagacttg acctgatagt
12481 ttggctgtga gcaattatgt gcttagtgca tctaatcgct tgagttaacg ccggcgaagc
12541 ggcgtcggct tgaacgaatt tctagctaga cattatttgc cgactacctt ggtgatctcg
12601 cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
12661 tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg
12721 cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
12781 taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc
12841 gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga
12901 tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
12961 agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca
13021 ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
13081 tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa
13141 gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg
13201 gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
13261 ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc
13321 tctgatagtt gagtcgatac ttcggcgatc accgcttccc ccatgatgtt taactttgtt
13381 ttagggcgac tgccctgctg cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc
13441 acggcgtaac gcgcttgctg cttggatgcc cgaggcatag actgtacccc aaaaaaacat
13501 gtcataacaa gaagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
13561 ggttctggac cagttgcgtg acggcagtta cgctacttgc attacagctt acgaaccgaa
13621 cgaggcttat gtccactggg ttcgtgcccg aattgatcac aggcagcaac gctctgtcat
13681 cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc ggcagcttag
13741 ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt acaacggctc
13801 tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat tttgtgccga
13861 gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt gtaaacaaat
13921 tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga attaacgccg
13981 aattgaatta tcagcttgca tgccggtcga tctagtaaca tagatgacac cgcgcgcgat
14041 aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc
14101 gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt
14161 acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga
14221 ttcaatctta agaaacttta ttgccaaatg tttgaacgat ctgcttgact ctagctagag
14281 tccgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
14341 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
14401 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
14461 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
14521 caggcatcgc cgtgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg
14581 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
14641 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
14701 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
14761 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
14821 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
14881 gtggccagcc acgatagccg cgctgcctcg tcttggagtt cattcagggc accggacagg
14941 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
15001 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
15061 ggagaacctg cgtgcaatcc atcttgttca atcatgcctc gatcgagttg agagtgaata
15121 tgagactcta attggatacc gaggggaatt tatggaacgt cagtggagca tttttgacaa
15181 gaaatatttg ctagctgata gtgaccttag gcgacttttg aacgcgcaat aatggtttct
15241 gacgtatgtg cttagctcat taaactccag aaacccgcgg ctgagtggct ccttcaacgt
15301 tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc gggggtcata
15361 acgtgactcc cttaattctc atgtatgata attcgagggt acccggggat cct
//
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：pK7GWIWG2II-RedRoot植物表达质粒
别称: pK7GWIWG2II-RedRoot
| 启动子: | 35s |
|---|---|
| 复制子: | pVS1V |
| 终止子: | NOS |
| 质粒分类: | 植物系列，gateway载体 |
| 质粒大小: | 15413bp |
| 质粒标签: | ccdB |
| 原核抗性: | Kan |
| 筛选标记: | G418 |
| 克隆菌株: | DB3.1 |
| 培养条件: | 37度 |
| 表达宿主: | 植物细胞 |
| 5'测序引物: | 35S:GACGCACAATCCCACTATCC |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | GateWay |
| 原核抗性： | Kan |
| 筛选标记： | Neo/G418 |
| 荧光蛋白： |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 15413 bp ds-DNA circular SYN 20-JUL-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pK7GWIWG2 II-RedRoot
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15413)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 19, 2016 from SnapGene Viewer 3.2.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..15413
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 65..317
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 2772..2896
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR1"
/note="recombination site for the Gateway(R) LR reaction"
promoter 2921..2951
/note="lac UV5 promoter"
/note="E. coli lac promoter with an ""up"" mutation"
CDS 3303..3608
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="ccdB"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(3649..3773)
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR2"
/note="recombination site for the Gateway(R) LR reaction"
CDS 4148..4807
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/note="CmR"
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
protein_bind 5210..5334
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR2"
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(5375..5680)
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="ccdB"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
promoter complement(6032..6062)
/note="lac UV5 promoter"
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind complement(6087..6211)
/gene="mutant version of attR"
/bound_moiety="LR Clonase(TM)"
/note="attR1"
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(6320..6664)
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 7357..7381
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 8681..9310
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 9744..10811
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 RepA"
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 10877..11071
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 11415..11555
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(11741..12329)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12573..13364)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/note="SmR"
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_feature 13892..13916
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
terminator 14009..14261
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(14301..15095)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(15129..15308)
/note="NOS promoter"
/note="nopaline synthase promoter"
ORIGIN
1 ctagatgcat gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttgggc
61 ccccgatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat
121 attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat
181 ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga
241 aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc
301 caaatgtttg aacgatcggg aattggatcc tacaggaaca ggtggtggcg gccctcggtg
361 cgctcgtact gctccacgat ggtgtagtcc tcgtttgtgg gaggtgatgt ccagcttgga
421 gtccacgtag tagtagccgg gcagctgcac gggcttcttg gccatgtaga tggacttgaa
481 ctccaccagg tagtggccgc cgtccttcag cttcagggcc ttgtggatct cgcccttcag
541 cacgccgtcg cgggggtaca ggcgctcggt ggaggcctcc cagcccatgg tcttcttctg
601 cattacgggg ccgtcggagg ggaagttcac gccgatgaac ttcaccttgt agatgaagca
661 gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat
721 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc ttgtagtcgg ggatgtcggc
781 ggggtgcttc acgtacacct tggagccgta ctggaactgg ggggacagga tgtcccaggc
841 gaagggcagg gggccgccct tggtcacctt cagcttcacg gtgttgtggc cctcgtaggg
901 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggtgc cctccatgcg
961 caccttgaag cgcatgaact ccttgatgac gttcttggag gagcgcgcca tggcaaagat
1021 ctgcatctgt taatcagaaa aactcagatt aatcgacaaa ttcgatcgca caaactagaa
1081 actaacacca gatctagata gaaatcacaa atcgaagagt aattattcga caaaactcaa
1141 attatttgaa caaatcggat gatatctatg aaaccctaat cgagaattaa gatgatatct
1201 aacgatcaaa cccagaaaat cgtcttcgat ctaagattaa cagaatctaa accaaagaac
1261 atatacgaaa ttgggatcga acgaaaacaa aatcgaagat tttgagagaa taaggaacac
1321 agaaatttac cttgatcacg gtagagagaa ttgagagaaa gtttttaaga ttttgagaaa
1381 ttgaaatctg aattgtgaag aagaagagct ctttgggtat tgttttatag aagaagaaga
1441 agaaaagacg aggacgacta ggtcacgaga aagctaaggc ggtgaagcaa tagctaataa
1501 taaaatgaca cgtgtattga gcgttgttta cacgcaaagt tgtttttggc taattgcctt
1561 atttttaggt tgaggaaaag tatttgtgct ttgagttgat aaacacgact cgtgtgtgcc
1621 ggctgcaacc actttgacgc cgtttattac tggacttcgt ccgacaacca caatttcctt
1681 aacggtcgtc ataagatcca gccgttgaga tttaacgatc gttacgattt atattttttt
1741 agcattatcg ttttattttt taaatatacg gtggagctga aaattggcaa taattgaacc
1801 gtgggtccca ctgcattgaa gcgtatttcg tattttctag aattcttcgt gctttatttc
1861 ttttcctttt tgtttttttt tgccatttat ctaatgcaag tgggcttata aaatcagtga
1921 atttcttgga aaagtaactt ctttatcgta taacatattg tgaaattatc catttctttt
1981 aattttttta gtgttattgg atatttttgt atgattattg atttgcatag gataatgact
2041 tttgtatcaa gttggtgaac aagtctcgtt aaaaaaggca agtggtttgg tgactcgatt
2101 tattcttgtt atttaattca tatatcaatg gatcttattt ggggcctggt ccatatttaa
2161 cactcgtgtt cagtccaatg accaataata ttttttcatt aataacaatg taacaagaat
2221 gatacacaaa acattctttg aataagttcg ctatgaagaa gggaacttat ccggtcctag
2281 atcatcagtt catacaaacc tccatagagt tcaacatctt aaacaagaat atcctgatct
2341 gaagaatgtg gaggctttag tcccttggat acttgggagg ctgtggaaga acagaaacga
2401 gctggtgctt aaagggaggg aatttggaac caatgaggta ttagtaagga cacaagaaga
2461 tgcagatgag tggattagaa ggaaagaggc tcagaatgta aggaaagcac caaccacaac
2521 gacgtcgcat gcctgcaggt cactggattt tggttttagg aattagaaat tttattgata
2581 gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg
2641 aaaccctata agaaccctaa ttcccttatc tgggaactac tcacacatta ttctggagaa
2701 aaatagagag agatagattt gtagagagag actggtgatt tttgcggact ctagcatggc
2761 cgcgggatat cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata
2821 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
2881 atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg
2941 ctcgtataat gtgtggattt tgagttagga tccggcttac taaaagccag ataacagtat
3001 gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt
3061 atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt gacagttgac agcgacagct
3121 atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca
3181 gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc
3241 tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca gggactggtg
3301 aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
3361 tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac
3421 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
3481 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
3541 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
3601 gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacca tagtgactgg
3661 atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata
3721 ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtgatatca
3781 ctagtgcggc cgcctgcagg tcgaccatat ggtcgacctg caggcggccg cactagtgat
3841 gctgttatgt tcagtgtcaa gctgacctgc aaacacgtta aatgctaaga agttagaata
3901 tatgagacac gttaactggt atatgaataa gctgtaaata accgagtata aactcattaa
3961 ctaatatcac ctctagagta taatataatc aaattcgaca atttgacttt caagagtagg
4021 ctaatgtaaa atctttatat atttctacaa tgttcaaaga aacagttgca tctaaacccc
4081 tatggccatc aaattcaatg aacgctaagc tgatccggcg agattttcag gagctaagga
4141 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
4201 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
4261 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
4321 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
4381 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
4441 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
4501 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
4561 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
4621 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
4681 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tctgtgatgg
4741 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
4801 ggcgtaaacg cgtggatcag cttaatatga ctctcaataa agtctcatac caacaagtgc
4861 caccttattc aaccatcaag aaaaaagcca aaatttatgc tactctaagg aaaacttcac
4921 taaagaagac gatttagagt gttttaccaa gaatttctgt catcttacta aacaactaaa
4981 gatcggtgtg atacaaaacc taatctcatt aaagtttatg ctaaaataag cataatttta
5041 cccactaagc gtgaccagat aaacataact cagcacacca gagcatatat attggtggct
5101 caaatcatag aaacttacag tgaagacaca gaaagccgta agaagaggca agagtatgaa
5161 accttacctc atcatttcca tgaggttgct tctgatcccg cgggatatca ccactttgta
5221 caagaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
5281 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggtcgac
5341 ctgcagactg gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa
5401 tggcgttttt gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg
5461 agaccggcac actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca
5521 ccaccgggta aagttcacgg gagactttat ctgacagcag acgtgcactg gccaggggga
5581 tcaccatccg tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat
5641 aacggctctc tcttttatag gtgtaaacct taaactgcat ttcaccagtc cctgttctcg
5701 tcagcaaaag agccgttcat ttcaataaac cgggcgacct cagccatccc ttcctgattt
5761 tccgctttcc agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc
5821 agaccggaga tattgacatc atatatgcct tgagcaactg atagctgtcg ctgtcaactg
5881 tcactgtaat acgctgcttc atagcacacc tctttttgac atacttcggg tatacatatc
5941 agtatatatt cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt
6001 tagtaagccg gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta
6061 aagcctgggg tgcctaatgc ggccgccata gtgactggat atgttgtgtt ttacagtatt
6121 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
6181 tttctcgttc agcttttttg tacaaacttg tgatatcact agtgcggccg cctgcaggtc
6241 gactagaata gtaaattgta atgttgtttg ttgtttgttt tgttgtggta attgttgtaa
6301 aaatacggat cgtcctgcag tcctctccaa atgaaatgaa cttccttata tagaggaagg
6361 gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg tcagtggaga tatcacatca
6421 atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg
6481 tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt
6541 atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg
6601 acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt
6661 ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt
6721 gtcgtgctcc accatgttga cgaagatttt cttcttgtca ttgagtcgta aaagactctg
6781 tatgaactgt tcgccagtct tcacggcgag ttctgttaga tcctcgatct gaatttttga
6841 ctccatggcc tttgattcag taggaactac tttcttagag actccaatct ctattacttg
6901 ccttggttta tgaagcaagc cttgaatcgt ccatactgga atagtacttc tgatcttgag
6961 aaatatatct ttctctgtgt tcttgatgca gttagtcctg aatcttttga ctgcatcttt
7021 aaccttcttg ggaaggtatt tgatctcctg gagattatta ctcgggtaga tcgtcttgat
7081 gagacctgcc gcgtaggcct ctctaaccat ctgtgggtca gcattctttc tgaaattgaa
7141 gaggctaatc ttctcattat cggtggtgaa catggtatcg tcaccttctc cgtcgaactt
7201 tcttcctaga tcgtagagat agagaaagtc gtccatggtg atctccgggg caaaggagat
7261 cagcttggct ctagtcgacc atatgggaga gctcaagctt agcttgagct tggatcagat
7321 tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa
7381 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta
7441 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact
7501 ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct
7561 tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt
7621 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac
7681 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc
7741 agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc
7801 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg
7861 cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc
7921 agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt
7981 agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg
8041 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc
8101 gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag
8161 atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca
8221 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg
8281 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc
8341 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc
8401 aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg
8461 tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt
8521 ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc
8581 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata
8641 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact
8701 taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca
8761 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg
8821 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga
8881 ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc
8941 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc
9001 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga
9061 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg
9121 catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg
9181 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc
9241 agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa
9301 actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc
9361 ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca
9421 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac
9481 gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca
9541 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat
9601 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg
9661 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc
9721 caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg
9781 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca
9841 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag
9901 aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg
9961 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca
10021 tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc
10081 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg
10141 tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat
10201 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac
10261 tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca
10321 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc
10381 tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa
10441 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag
10501 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca
10561 tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt
10621 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca
10681 ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg
10741 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg
10801 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc
10861 gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga
10921 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag
10981 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta
11041 aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc
11101 tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc
11161 ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc
11221 gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc
11281 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
11341 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
11401 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
11461 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
11521 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
11581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
11641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
11701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
11761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
11821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
11881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
11941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
12001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
12061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
12121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
12181 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
12241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
12301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
12361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcatgat atatctccca
12421 atttgtgtag ggcttattat gcacgcttaa aaataataaa agcagacttg acctgatagt
12481 ttggctgtga gcaattatgt gcttagtgca tctaatcgct tgagttaacg ccggcgaagc
12541 ggcgtcggct tgaacgaatt tctagctaga cattatttgc cgactacctt ggtgatctcg
12601 cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
12661 tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg
12721 cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
12781 taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc
12841 gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga
12901 tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
12961 agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca
13021 ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
13081 tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa
13141 gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg
13201 gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
13261 ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc
13321 tctgatagtt gagtcgatac ttcggcgatc accgcttccc ccatgatgtt taactttgtt
13381 ttagggcgac tgccctgctg cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc
13441 acggcgtaac gcgcttgctg cttggatgcc cgaggcatag actgtacccc aaaaaaacat
13501 gtcataacaa gaagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
13561 ggttctggac cagttgcgtg acggcagtta cgctacttgc attacagctt acgaaccgaa
13621 cgaggcttat gtccactggg ttcgtgcccg aattgatcac aggcagcaac gctctgtcat
13681 cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc ggcagcttag
13741 ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt acaacggctc
13801 tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat tttgtgccga
13861 gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt gtaaacaaat
13921 tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga attaacgccg
13981 aattgaatta tcagcttgca tgccggtcga tctagtaaca tagatgacac cgcgcgcgat
14041 aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc
14101 gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt
14161 acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga
14221 ttcaatctta agaaacttta ttgccaaatg tttgaacgat ctgcttgact ctagctagag
14281 tccgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
14341 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
14401 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
14461 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
14521 caggcatcgc cgtgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg
14581 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
14641 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
14701 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
14761 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
14821 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
14881 gtggccagcc acgatagccg cgctgcctcg tcttggagtt cattcagggc accggacagg
14941 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
15001 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
15061 ggagaacctg cgtgcaatcc atcttgttca atcatgcctc gatcgagttg agagtgaata
15121 tgagactcta attggatacc gaggggaatt tatggaacgt cagtggagca tttttgacaa
15181 gaaatatttg ctagctgata gtgaccttag gcgacttttg aacgcgcaat aatggtttct
15241 gacgtatgtg cttagctcat taaactccag aaacccgcgg ctgagtggct ccttcaacgt
15301 tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc gggggtcata
15361 acgtgactcc cttaattctc atgtatgata attcgagggt acccggggat cct
//
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
| P3090/pENTR223-CEP72（568-1944bp）人源基因质粒 |
| P3091/pENTR223-INPP5K人源基因质粒 |
| P3092/pENTR223-CCKBR（1同义突变）人源基因质粒 |
| P3093/pENTR223-DACH1(580-2121bp）人源基因质粒 |
| P3094/pENTR223-THOC1（1点突变1同义突变）人源基因质粒 |
| P3095/pENTR223-SLCO6A1（有一同义突变）人源基因质粒 |
| P3096/pENTR223-GPKOW人源基因质粒 |
| P3097/pENTR223-FANCM人源基因质粒 |
| P3098/pENTR223-C1R-C455T人源基因质粒 |
| P3099/pENTR223-NVL（319-2298bp1同义突变）人源基因质粒 |
| P3100/pENTR223-DBF4（2点突变）人源基因质粒 |
| P3101/pENTR223-GPC-PD1人源基因质粒 |
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文献和实验相关实验
原核表达载体 pQE80L 大肠杆菌质粒 Amp pCold I 大肠杆菌质粒 Amp 冷启动表达载体，His标签 pCold II 大肠杆菌质粒 Amp 冷启动表达载体，His标签
。 利用基因工程技术表达外源蛋白。其产量还不高，难以满足大规模的实际应用。通过转基因动物或转基因植物技术可从动物的乳汁或植物的叶组织中很方便地获得大量较纯的生物活性物质，但目前这项技术还不很成熟，有待进一步研究。 4.结语 目前已经建立了多种诱导表达系统，但它们都有其优点和不足，这就需要我们根据自己的要求选用适当的表达系统。一般来说，一个理想的可诱导表达系统需要符合下述几个方面的要求： ①特异性：该系统不受其他内源因素的影响，仅能被外源的非毒性药物所活化
…Actin…BamH1…SV40 poly A+ 4. pSV2表达载体 特点：该表达质粒是以病责SV40启动子驱动在真核细胞目的基因进行表达的，克隆位点为Hind111。SV40启动子具有组织/细胞的选择特异性。此载体不含neo基因，故不能用来筛选、建立稳定的表达细胞株。 图示为启动子和多克隆位点区域： Pvu11…pSV40….Hind111….SV40 IVS….SV40 polyA+ 5. CMV4 表达载体 特点:该真核
pK7GWIWG2II-RedRoot植物表达质粒
￥100 - 1000
