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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：
pRGEB31-gRNA
- 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：pRGEB31-未知gRNA
别称: pRGEB31-gRNA
复制子: pVS1
原核抗性: Kan
筛选标记：Hyg
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主：植物
载体用途：基因敲除
基因种属：
基因类型：CRISPR
原核抗性：Kan
真核抗性：Hyg
荧光蛋白：
质粒简介
pRGEB31-未知gRNA质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 15041 bp ds-DNA circular SYN 17-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..15041
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 6..386
/label=OsU3 promoter
/note="Oryza sativa (rice) snRNA U3 promoter"
misc_feature 387..406
/label=δ֪gRNA
misc_RNA 407..482
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 1048..1393
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 1484..1549
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1556..1576
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1601..5701
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5702..5749
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
protein_bind complement(5782..5806)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
terminator 5862..6114
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 6359..6383
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 7683..8312
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 8746..9813
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 9879..10073
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 10417..10557
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(10743..11331)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11418..12212)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 12637..12661
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 12739..12913
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(12953..13978)
/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
K"
promoter complement(14046..14723)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
ORIGIN
1 agcttaagga atctttaaac atacgaacag atcacttaaa gttcttctga agcaacttaa
61 agttatcagg catgcatgga tcttggagga atcagatgtg cagtcaggga ccatagcaca
121 agacaggcgt cttctactgg tgctaccagc aaatgctgga agccgggaac actgggtacg
181 ttggaaacca cgtgatgtga agaagtaaga taaactgtag gagaaaagca tttcgtagtg
241 ggccatgaag cctttcagga catgtattgc agtatgggcc ggcccattac gcaattggac
301 gacaacaaag actagtatta gtaccacctc ggctatccac atagatcaaa gctgatttaa
361 aagagttgtg cagatgatcc gtggcacaac tggcatgcga accaacgttt tagagctaga
421 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
481 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
541 gtgcatgcct gcaggtcccc agattagcct tttcaatttc agaaagaatg ctaacccaca
601 gatggttaga gaggcttacg cagcagcact catcaagacg atctacccga gcaataatct
661 ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa
721 ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg
781 gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa
841 ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga
901 actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa
961 gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga
1021 tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa
1081 cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga
1141 aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc
1201 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga
1261 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga
1321 tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca
1381 tttggagaga acacggggga ctctagagtt atcaacaagt ttgtacaaaa aagcaggctt
1441 cgaaggagat agaaccaatt ctctaaggaa atacttaacc atggactata aggaccacga
1501 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
1561 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
1621 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
1681 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
1741 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
1801 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
1861 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
1921 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
1981 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
2041 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
2101 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
2161 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
2221 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
2281 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
2341 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
2401 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
2461 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
2521 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
2581 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
2641 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
2701 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
2761 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
2821 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
2881 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
2941 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
3001 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
3061 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
3121 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
3181 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
3241 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
3301 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
3361 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
3421 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
3481 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
3541 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
3601 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
3661 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
3721 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
3781 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
3841 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
3901 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
3961 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
4021 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
4081 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
4141 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
4201 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
4261 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
4321 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
4381 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
4441 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
4501 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
4561 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
4621 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
4681 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
4741 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
4801 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
4861 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
4921 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
4981 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
5041 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
5101 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
5161 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
5221 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
5281 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
5341 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
5401 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
5461 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
5521 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
5581 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
5641 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
5701 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcgc
5761 ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggttgat aacagcgact
5821 acaaggatga cgatgacaag gcttagagct cgaatttccc cgatcgttca aacatttggc
5881 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
5941 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
6001 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
6061 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat
6121 tcactggccg tcgttttaca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
6181 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
6241 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgctaga
6301 gcagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta tcagtgtttg
6361 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt
6421 taaaagggcg tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg
6481 ttcccctcgg gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt
6541 ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg
6601 acaggctgcc gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa
6661 gtagaatact tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg
6721 cctgctgggc tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga
6781 actgcacgcg gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga
6841 ccgcccggag ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac
6901 caggctagac cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca
6961 ggaggccggc gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc
7021 cggccgcatg gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat
7081 cgaccgcacc cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg
7141 ccctaccctc accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg
7201 caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact
7261 tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga
7321 cgcattgacc gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc
7381 atgaaaccgc accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg
7441 gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg
7501 catgaaatcc tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc
7561 gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg
7621 cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac
7681 gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa
7741 cccatctagc ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc
7801 cccagggcag tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg
7861 gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag
7921 tgatcgacgg agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact
7981 tcgtgctgat tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg
8041 agctggttaa gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt
8101 cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg
8161 agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg
8221 ccggcacaac cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc
8281 tggccgctga aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa
8341 aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt
8401 ggccagcctg gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga
8461 tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc
8521 tgaatacatc gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt
8581 tagcggctaa aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat
8641 gccccatgtg tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc
8701 cctgcaatgg cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc
8761 ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg
8821 caggccgccc agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg
8881 gccgctgatc gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt
8941 aggaagccgc ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg
9001 ggcacccgcg atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac
9061 cgacgagctg gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca
9121 gggccggccg gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat
9181 ctaaccgaat ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc
9241 cgtccacacg ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa
9301 gacgacctgg tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg
9361 aagaaggcca agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc
9421 tacaagatcg taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat
9481 tggatgtacc gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat
9541 tactttttga tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca
9601 ggcaaggcag aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga
9661 gagttcaaga agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag
9721 tacgatttga aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac
9781 ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt
9841 gccctagcag gggaaaaagg tcgaaaagca ctctttcctg tggatagcac gtacattggg
9901 aacccaaagc cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt
9961 gggaaccggt cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc
10021 taaaactctt taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct
10081 ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta
10141 cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta
10201 cggccaggca atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc
10261 ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
10321 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
10381 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
10441 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
10501 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
10561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
10621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
10681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
10741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
10801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
10861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
10921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
10981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
11041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
11101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
11161 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
11221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
11281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
11341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
11401 gtcatgcatt ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc
11461 caatcaggct tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata
11521 tcctccctga tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt
11581 ctcccaagat caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc
11641 aggtcgccgt gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac
11701 agctcgcgcg gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc
11761 agatcgttat tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag
11821 ggacaatccg atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata
11881 atcttttcag ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca
11941 ctcatgagca gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc
12001 agctttcctt ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct
12061 ttataccggc tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat
12121 accttagcag gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt
12181 ttcaattccg gtgatattct cattttagcc atttattatt tccttcctct tttctacagt
12241 atttaaagat accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg
12301 cattctaaaa ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt
12361 ataacatagt atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct
12421 gtcatcgtta caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag
12481 cttagttgcc gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac
12541 ggctctcccg ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt
12601 gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa
12661 caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa
12721 cgccgaatta attcggggga tctggatttt agtactggat tttggtttta ggaattagaa
12781 attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc
12841 aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact actcacacat
12901 tattatggag aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct
12961 ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc
13021 catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc
13081 cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt
13141 caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg
13201 gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag
13261 ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg
13321 cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc
13381 cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct
13441 catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat
13501 acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc
13561 cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat
13621 ccatagcctc cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca
13681 acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa
13741 tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat
13801 ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat
13861 cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt
13921 cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat
13981 ctcattgccc cccgggatct gcgaaagctc gagagagata gatttgtaga gagagactgg
14041 tgatttcagc gtgtcctctc caaatgaaat gaacttcctt atatagagga aggtcttgcg
14101 aaggatagtg ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt
14161 gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc
14221 catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat
14281 gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag
14341 ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag
14401 ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct
14461 ccaccatgtt atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt
14521 ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt
14581 gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta
14641 ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata
14701 ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat
14761 tcttggagta gacgagagtg tcgtgctcca ccatgttggc aagctgctct agccaatacg
14821 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
14881 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
14941 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
15001 acaatttcac acaggaaaca gctatgacca tgattacgcc a
//
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：pRGEB31-未知gRNA
别称: pRGEB31-gRNA
复制子: pVS1
原核抗性: Kan
筛选标记：Hyg
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主：植物
载体用途：基因敲除
基因种属：
基因类型：CRISPR
原核抗性：Kan
真核抗性：Hyg
荧光蛋白：
质粒简介
pRGEB31-未知gRNA质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 15041 bp ds-DNA circular SYN 17-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..15041
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 6..386
/label=OsU3 promoter
/note="Oryza sativa (rice) snRNA U3 promoter"
misc_feature 387..406
/label=δ֪gRNA
misc_RNA 407..482
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 1048..1393
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 1484..1549
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1556..1576
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1601..5701
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5702..5749
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
protein_bind complement(5782..5806)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
terminator 5862..6114
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 6359..6383
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 7683..8312
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 8746..9813
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 9879..10073
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 10417..10557
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(10743..11331)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11418..12212)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 12637..12661
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 12739..12913
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(12953..13978)
/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
K"
promoter complement(14046..14723)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
ORIGIN
1 agcttaagga atctttaaac atacgaacag atcacttaaa gttcttctga agcaacttaa
61 agttatcagg catgcatgga tcttggagga atcagatgtg cagtcaggga ccatagcaca
121 agacaggcgt cttctactgg tgctaccagc aaatgctgga agccgggaac actgggtacg
181 ttggaaacca cgtgatgtga agaagtaaga taaactgtag gagaaaagca tttcgtagtg
241 ggccatgaag cctttcagga catgtattgc agtatgggcc ggcccattac gcaattggac
301 gacaacaaag actagtatta gtaccacctc ggctatccac atagatcaaa gctgatttaa
361 aagagttgtg cagatgatcc gtggcacaac tggcatgcga accaacgttt tagagctaga
421 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
481 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
541 gtgcatgcct gcaggtcccc agattagcct tttcaatttc agaaagaatg ctaacccaca
601 gatggttaga gaggcttacg cagcagcact catcaagacg atctacccga gcaataatct
661 ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa
721 ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg
781 gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa
841 ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga
901 actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa
961 gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga
1021 tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa
1081 cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga
1141 aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc
1201 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga
1261 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga
1321 tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca
1381 tttggagaga acacggggga ctctagagtt atcaacaagt ttgtacaaaa aagcaggctt
1441 cgaaggagat agaaccaatt ctctaaggaa atacttaacc atggactata aggaccacga
1501 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
1561 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
1621 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
1681 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
1741 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
1801 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
1861 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
1921 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
1981 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
2041 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
2101 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
2161 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
2221 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
2281 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
2341 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
2401 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
2461 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
2521 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
2581 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
2641 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
2701 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
2761 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
2821 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
2881 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
2941 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
3001 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
3061 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
3121 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
3181 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
3241 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
3301 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
3361 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
3421 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
3481 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
3541 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
3601 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
3661 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
3721 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
3781 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
3841 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
3901 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
3961 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
4021 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
4081 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
4141 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
4201 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
4261 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
4321 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
4381 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
4441 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
4501 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
4561 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
4621 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
4681 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
4741 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
4801 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
4861 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
4921 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
4981 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
5041 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
5101 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
5161 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
5221 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
5281 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
5341 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
5401 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
5461 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
5521 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
5581 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
5641 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
5701 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcgc
5761 ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggttgat aacagcgact
5821 acaaggatga cgatgacaag gcttagagct cgaatttccc cgatcgttca aacatttggc
5881 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
5941 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
6001 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
6061 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat
6121 tcactggccg tcgttttaca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
6181 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
6241 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgctaga
6301 gcagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta tcagtgtttg
6361 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt
6421 taaaagggcg tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg
6481 ttcccctcgg gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt
6541 ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg
6601 acaggctgcc gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa
6661 gtagaatact tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg
6721 cctgctgggc tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga
6781 actgcacgcg gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga
6841 ccgcccggag ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac
6901 caggctagac cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca
6961 ggaggccggc gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc
7021 cggccgcatg gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat
7081 cgaccgcacc cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg
7141 ccctaccctc accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg
7201 caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact
7261 tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga
7321 cgcattgacc gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc
7381 atgaaaccgc accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg
7441 gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg
7501 catgaaatcc tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc
7561 gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg
7621 cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac
7681 gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa
7741 cccatctagc ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc
7801 cccagggcag tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg
7861 gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag
7921 tgatcgacgg agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact
7981 tcgtgctgat tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg
8041 agctggttaa gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt
8101 cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg
8161 agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg
8221 ccggcacaac cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc
8281 tggccgctga aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa
8341 aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt
8401 ggccagcctg gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga
8461 tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc
8521 tgaatacatc gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt
8581 tagcggctaa aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat
8641 gccccatgtg tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc
8701 cctgcaatgg cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc
8761 ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg
8821 caggccgccc agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg
8881 gccgctgatc gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt
8941 aggaagccgc ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg
9001 ggcacccgcg atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac
9061 cgacgagctg gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca
9121 gggccggccg gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat
9181 ctaaccgaat ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc
9241 cgtccacacg ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa
9301 gacgacctgg tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg
9361 aagaaggcca agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc
9421 tacaagatcg taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat
9481 tggatgtacc gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat
9541 tactttttga tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca
9601 ggcaaggcag aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga
9661 gagttcaaga agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag
9721 tacgatttga aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac
9781 ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt
9841 gccctagcag gggaaaaagg tcgaaaagca ctctttcctg tggatagcac gtacattggg
9901 aacccaaagc cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt
9961 gggaaccggt cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc
10021 taaaactctt taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct
10081 ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta
10141 cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta
10201 cggccaggca atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc
10261 ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
10321 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
10381 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
10441 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
10501 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
10561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
10621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
10681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
10741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
10801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
10861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
10921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
10981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
11041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
11101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
11161 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
11221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
11281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
11341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
11401 gtcatgcatt ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc
11461 caatcaggct tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata
11521 tcctccctga tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt
11581 ctcccaagat caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc
11641 aggtcgccgt gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac
11701 agctcgcgcg gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc
11761 agatcgttat tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag
11821 ggacaatccg atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata
11881 atcttttcag ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca
11941 ctcatgagca gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc
12001 agctttcctt ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct
12061 ttataccggc tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat
12121 accttagcag gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt
12181 ttcaattccg gtgatattct cattttagcc atttattatt tccttcctct tttctacagt
12241 atttaaagat accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg
12301 cattctaaaa ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt
12361 ataacatagt atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct
12421 gtcatcgtta caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag
12481 cttagttgcc gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac
12541 ggctctcccg ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt
12601 gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa
12661 caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa
12721 cgccgaatta attcggggga tctggatttt agtactggat tttggtttta ggaattagaa
12781 attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc
12841 aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact actcacacat
12901 tattatggag aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct
12961 ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc
13021 catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc
13081 cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt
13141 caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg
13201 gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag
13261 ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg
13321 cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc
13381 cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct
13441 catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat
13501 acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc
13561 cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat
13621 ccatagcctc cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca
13681 acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa
13741 tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat
13801 ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat
13861 cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt
13921 cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat
13981 ctcattgccc cccgggatct gcgaaagctc gagagagata gatttgtaga gagagactgg
14041 tgatttcagc gtgtcctctc caaatgaaat gaacttcctt atatagagga aggtcttgcg
14101 aaggatagtg ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt
14161 gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc
14221 catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat
14281 gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag
14341 ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag
14401 ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct
14461 ccaccatgtt atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt
14521 ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt
14581 gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta
14641 ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata
14701 ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat
14761 tcttggagta gacgagagtg tcgtgctcca ccatgttggc aagctgctct agccaatacg
14821 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
14881 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
14941 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
15001 acaatttcac acaggaaaca gctatgacca tgattacgcc a
//
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
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