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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：
pCAMBIA1303
- 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：pCAMBIA1303植物表达质粒
别称: pCAMBIA1303
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 12361 bp ds-DNA circular SYN 14-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12361)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Wednesday, September 14, 2016 from SnapGene Viewer 3.2.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12361
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 20..1825
/codon_start=1
/gene="uidA (or gusA) from E. coli"
/product="beta-glucuronidase"
/note="GUS"
/translation="LRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAIA
VPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEVM
EHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHDF
FNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQV
VATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGQQFL
INHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDWA
DEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARDK
NHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISDL
FDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYT
DMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKP
KSAAFLLQKRWTGMNFGEKPQQGGKQ"
CDS 1829..2539
/codon_start=1
/gene="mgfp5"
/product="GFP with folding enhancement mutations"
/note="mgfp5"
/note="suitable for expression in plants due to removal of
a cryptic intron"
/translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
VTAAGITHGMDELYK"
CDS 2546..2563
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2595..2847
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2869..2893
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 4193..4822
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 5256..6323
/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 6389..6583
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6927..7067
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(7253..7841)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7928..8722)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 9147..9171
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 9249..9423
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(9463..10488)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(10556..11233)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 11424..11445
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11460..11490
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 11498..11514
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 11522..11538
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 11534..11767
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 11548..11604
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(11608..11624)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 12001..12346
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
ORIGIN
1 catggtagat ctgactagtt tacgtcctgt agaaacccca acccgtgaaa tcaaaaaact
61 cgacggcctg tgggcattca gtctggatcg cgaaaactgt ggaattgatc agcgttggtg
121 ggaaagcgcg ttacaagaaa gccgggcaat tgctgtgcca ggcagtttta acgatcagtt
181 cgccgatgca gatattcgta attatgcggg caacgtctgg tatcagcgcg aagtctttat
241 accgaaaggt tgggcaggcc agcgtatcgt gctgcgtttc gatgcggtca ctcattacgg
301 caaagtgtgg gtcaataatc aggaagtgat ggagcatcag ggcggctata cgccatttga
361 agccgatgtc acgccgtatg ttattgccgg gaaaagtgta cgtatcaccg tttgtgtgaa
421 caacgaactg aactggcaga ctatcccgcc gggaatggtg attaccgacg aaaacggcaa
481 gaaaaagcag tcttacttcc atgatttctt taactatgcc ggaatccatc gcagcgtaat
541 gctctacacc acgccgaaca cctgggtgga cgatatcacc gtggtgacgc atgtcgcgca
601 agactgtaac cacgcgtctg ttgactggca ggtggtggcc aatggtgatg tcagcgttga
661 actgcgtgat gcggatcaac aggtggttgc aactggacaa ggcactagcg ggactttgca
721 agtggtgaat ccgcacctct ggcaaccggg tgaaggttat ctctatgaac tgtgcgtcac
781 agccaaaagc cagacagagt gtgatatcta cccgcttcgc gtcggcatcc ggtcagtggc
841 agtgaagggc caacagttcc tgattaacca caaaccgttc tactttactg gctttggtcg
901 tcatgaagat gcggacttac gtggcaaagg attcgataac gtgctgatgg tgcacgacca
961 cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc cttacgctga
1021 agagatgctc gactgggcag atgaacatgg catcgtggtg attgatgaaa ctgctgctgt
1081 cggctttcag ctgtctttag gcattggttt cgaagcgggc aacaagccga aagaactgta
1141 cagcgaagag gcagtcaacg gggaaactca gcaagcgcac ttacaggcga ttaaagagct
1201 gatagcgcgt gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca acgaaccgga
1261 tacccgtccg caaggtgcac gggaatattt cgcgccactg gcggaagcaa cgcgtaaact
1321 cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc tgcgacgctc acaccgatac
1381 catcagcgat ctctttgatg tgctgtgcct gaaccgttat tacggatggt atgtccaaag
1441 cggcgatttg gaaacggcag agaaggtact ggaaaaagaa cttctggcct ggcaggagaa
1501 actgcatcag ccgattatca tcaccgaata cggcgtggat acgttagccg ggctgcactc
1561 aatgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata tgtatcaccg
1621 cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta tggaatttcg ccgattttgc
1681 gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa gggatcttca ctcgcgaccg
1741 caaaccgaag tcggcggctt ttctgctgca aaaacgctgg actggcatga acttcggtga
1801 aaaaccgcag cagggaggca aacaagctag taaaggagaa gaacttttca ctggagttgt
1861 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
1921 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
1981 actacctgtt ccgtggccaa cacttgtcac tactttctct tatggtgttc aatgcttttc
2041 aagataccca gatcatatga agcggcacga cttcttcaag agcgccatgc ctgagggata
2101 cgtgcaggag aggaccatct tcttcaagga cgacgggaac tacaagacac gtgctgaagt
2161 caagtttgag ggagacaccc tcgtcaacag gatcgagctt aagggaatcg atttcaagga
2221 ggacggaaac atcctcggcc acaagttgga atacaactac aactcccaca acgtatacat
2281 catggccgac aagcaaaaga acggcatcaa agccaacttc aagacccgcc acaacatcga
2341 agacggcggc gtgcaactcg ctgatcatta tcaacaaaat actccaattg gcgatggccc
2401 tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa
2461 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
2521 catggatgaa ctatacaaag ctagccacca ccaccaccac cacgtgtgaa ttggtgacca
2581 gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
2641 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
2701 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
2761 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
2821 gcgcggtgtc atctatgtta ctagatcggg aattaaacta tcagtgtttg acaggatata
2881 ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt taaaagggcg
2941 tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg
3001 gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca
3061 gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc
3121 gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact
3181 tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc
3241 tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg
3301 gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag
3361 ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac
3421 cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc
3481 gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg
3541 gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc
3601 cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc
3661 accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa
3721 gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc
3781 gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc
3841 gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc
3901 accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg
3961 cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc
4021 tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa
4081 ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg
4141 tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt
4201 tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc
4261 ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag
4321 tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg
4381 cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg
4441 agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat
4501 tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa
4561 gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat
4621 caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat
4681 tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac
4741 cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga
4801 aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac
4861 acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg
4921 gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag
4981 ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc
5041 gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa
5101 aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg
5161 tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg
5221 cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc ggcccggtac
5281 aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc
5341 agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc
5401 gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc
5461 ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg
5521 atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg
5581 gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg
5641 gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat
5701 ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg
5761 ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg
5821 tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca
5881 agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg
5941 taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc
6001 gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga
6061 tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag
6121 aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga
6181 agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga
6241 aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg
6301 gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag
6361 gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc
6421 cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt
6481 cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt
6541 taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca
6601 cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg
6661 cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca
6721 atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag
6781 gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
6841 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
6901 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
6961 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
7021 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
7081 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
7141 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
7201 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
7261 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
7321 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
7381 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
7441 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
7501 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
7561 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
7621 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
7681 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
7741 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
7801 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
7861 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt
7921 ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct
7981 tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga
8041 tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat
8101 caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt
8161 gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg
8221 gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat
8281 tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg
8341 atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag
8401 ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca
8461 gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt
8521 ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc
8581 tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag
8641 gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg
8701 gtgatattct cattttagcc atttattatt tccttcctct tttctacagt atttaaagat
8761 accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa
8821 ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt
8881 atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta
8941 caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc
9001 gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg
9061 ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc
9121 cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg
9181 cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta
9241 attcggggga tctggatttt agtactggat tttggtttta ggaattagaa attttattga
9301 tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag
9361 cgaaacccta taggaaccct aattccctta tctgggaact actcacacat tattatggag
9421 aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct ttgccctcgg
9481 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
9541 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
9601 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
9661 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg gcgatcctgc
9721 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
9781 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
9841 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
9901 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
9961 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
10021 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
10081 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatagcctc
10141 cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
10201 ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa tgtcaagcac
10261 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
10321 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
10381 agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc
10441 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat ctcattgccc
10501 cccgggatct gcgaaagctc gagagagata gatttgtaga gagagactgg tgatttcagc
10561 gtgtcctctc caaatgaaat gaacttcctt atatagagga aggtcttgcg aaggatagtg
10621 ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga
10681 cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg
10741 accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt
10801 gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg
10861 gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc
10921 ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt
10981 atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct
11041 cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc
11101 ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt
11161 gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg
11221 ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta
11281 gacgagagtg tcgtgctcca ccatgttggc aagctgctct agccaatacg caaaccgcct
11341 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
11401 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
11461 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
11521 acaggaaaca gctatgacca tgattacgaa ttcgagctcg gtacccgggg atcctctaga
11581 gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
11641 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
11701 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
11761 atgctagagc agcttgagct tggatcagat tgtcgtttcc cgccttcagt ttagcttcat
11821 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
11881 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
11941 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
12001 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
12061 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
12121 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
12181 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
12241 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
12301 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactcttga
12361 c
//
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：pCAMBIA1303植物表达质粒
别称: pCAMBIA1303
| 复制子: | pVS1 |
|---|---|
| 质粒分类: | 植物系列，蛋白过表达载体 |
| 质粒大小: | 12361bp |
| 原核抗性: | Kan |
| 筛选标记: | Hyg |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 植物细胞 |
| 5'测序引物: | M13R:CAGGAAACAGCTATGACC |
| 3'测序引物: | M13F:TGTAAAACGACGGCCAGT |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Kan |
| 筛选标记： | Hyg |
| 荧光蛋白： | 绿色,Gus |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 12361 bp ds-DNA circular SYN 14-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12361)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Wednesday, September 14, 2016 from SnapGene Viewer 3.2.1
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12361
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 20..1825
/codon_start=1
/gene="uidA (or gusA) from E. coli"
/product="beta-glucuronidase"
/note="GUS"
/translation="LRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAIA
VPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEVM
EHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHDF
FNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQV
VATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGQQFL
INHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDWA
DEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARDK
NHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISDL
FDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYT
DMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKP
KSAAFLLQKRWTGMNFGEKPQQGGKQ"
CDS 1829..2539
/codon_start=1
/gene="mgfp5"
/product="GFP with folding enhancement mutations"
/note="mgfp5"
/note="suitable for expression in plants due to removal of
a cryptic intron"
/translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
VTAAGITHGMDELYK"
CDS 2546..2563
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2595..2847
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2869..2893
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 4193..4822
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 5256..6323
/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 6389..6583
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6927..7067
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(7253..7841)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7928..8722)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 9147..9171
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 9249..9423
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(9463..10488)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(10556..11233)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 11424..11445
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11460..11490
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 11498..11514
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 11522..11538
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 11534..11767
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 11548..11604
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(11608..11624)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 12001..12346
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
ORIGIN
1 catggtagat ctgactagtt tacgtcctgt agaaacccca acccgtgaaa tcaaaaaact
61 cgacggcctg tgggcattca gtctggatcg cgaaaactgt ggaattgatc agcgttggtg
121 ggaaagcgcg ttacaagaaa gccgggcaat tgctgtgcca ggcagtttta acgatcagtt
181 cgccgatgca gatattcgta attatgcggg caacgtctgg tatcagcgcg aagtctttat
241 accgaaaggt tgggcaggcc agcgtatcgt gctgcgtttc gatgcggtca ctcattacgg
301 caaagtgtgg gtcaataatc aggaagtgat ggagcatcag ggcggctata cgccatttga
361 agccgatgtc acgccgtatg ttattgccgg gaaaagtgta cgtatcaccg tttgtgtgaa
421 caacgaactg aactggcaga ctatcccgcc gggaatggtg attaccgacg aaaacggcaa
481 gaaaaagcag tcttacttcc atgatttctt taactatgcc ggaatccatc gcagcgtaat
541 gctctacacc acgccgaaca cctgggtgga cgatatcacc gtggtgacgc atgtcgcgca
601 agactgtaac cacgcgtctg ttgactggca ggtggtggcc aatggtgatg tcagcgttga
661 actgcgtgat gcggatcaac aggtggttgc aactggacaa ggcactagcg ggactttgca
721 agtggtgaat ccgcacctct ggcaaccggg tgaaggttat ctctatgaac tgtgcgtcac
781 agccaaaagc cagacagagt gtgatatcta cccgcttcgc gtcggcatcc ggtcagtggc
841 agtgaagggc caacagttcc tgattaacca caaaccgttc tactttactg gctttggtcg
901 tcatgaagat gcggacttac gtggcaaagg attcgataac gtgctgatgg tgcacgacca
961 cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc cttacgctga
1021 agagatgctc gactgggcag atgaacatgg catcgtggtg attgatgaaa ctgctgctgt
1081 cggctttcag ctgtctttag gcattggttt cgaagcgggc aacaagccga aagaactgta
1141 cagcgaagag gcagtcaacg gggaaactca gcaagcgcac ttacaggcga ttaaagagct
1201 gatagcgcgt gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca acgaaccgga
1261 tacccgtccg caaggtgcac gggaatattt cgcgccactg gcggaagcaa cgcgtaaact
1321 cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc tgcgacgctc acaccgatac
1381 catcagcgat ctctttgatg tgctgtgcct gaaccgttat tacggatggt atgtccaaag
1441 cggcgatttg gaaacggcag agaaggtact ggaaaaagaa cttctggcct ggcaggagaa
1501 actgcatcag ccgattatca tcaccgaata cggcgtggat acgttagccg ggctgcactc
1561 aatgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata tgtatcaccg
1621 cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta tggaatttcg ccgattttgc
1681 gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa gggatcttca ctcgcgaccg
1741 caaaccgaag tcggcggctt ttctgctgca aaaacgctgg actggcatga acttcggtga
1801 aaaaccgcag cagggaggca aacaagctag taaaggagaa gaacttttca ctggagttgt
1861 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
1921 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
1981 actacctgtt ccgtggccaa cacttgtcac tactttctct tatggtgttc aatgcttttc
2041 aagataccca gatcatatga agcggcacga cttcttcaag agcgccatgc ctgagggata
2101 cgtgcaggag aggaccatct tcttcaagga cgacgggaac tacaagacac gtgctgaagt
2161 caagtttgag ggagacaccc tcgtcaacag gatcgagctt aagggaatcg atttcaagga
2221 ggacggaaac atcctcggcc acaagttgga atacaactac aactcccaca acgtatacat
2281 catggccgac aagcaaaaga acggcatcaa agccaacttc aagacccgcc acaacatcga
2341 agacggcggc gtgcaactcg ctgatcatta tcaacaaaat actccaattg gcgatggccc
2401 tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa
2461 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
2521 catggatgaa ctatacaaag ctagccacca ccaccaccac cacgtgtgaa ttggtgacca
2581 gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
2641 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
2701 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
2761 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
2821 gcgcggtgtc atctatgtta ctagatcggg aattaaacta tcagtgtttg acaggatata
2881 ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt taaaagggcg
2941 tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg
3001 gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca
3061 gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc
3121 gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact
3181 tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc
3241 tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg
3301 gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag
3361 ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac
3421 cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc
3481 gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg
3541 gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc
3601 cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc
3661 accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa
3721 gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc
3781 gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc
3841 gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc
3901 accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg
3961 cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc
4021 tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa
4081 ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg
4141 tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt
4201 tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc
4261 ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag
4321 tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg
4381 cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg
4441 agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat
4501 tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa
4561 gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat
4621 caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat
4681 tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac
4741 cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga
4801 aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac
4861 acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg
4921 gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag
4981 ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc
5041 gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa
5101 aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg
5161 tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg
5221 cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc ggcccggtac
5281 aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc
5341 agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc
5401 gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc
5461 ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg
5521 atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg
5581 gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg
5641 gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat
5701 ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg
5761 ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg
5821 tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca
5881 agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg
5941 taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc
6001 gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga
6061 tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag
6121 aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga
6181 agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga
6241 aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg
6301 gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag
6361 gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc
6421 cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt
6481 cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt
6541 taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca
6601 cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg
6661 cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca
6721 atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag
6781 gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
6841 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
6901 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
6961 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
7021 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
7081 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
7141 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
7201 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
7261 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
7321 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
7381 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
7441 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
7501 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
7561 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
7621 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
7681 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
7741 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
7801 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
7861 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt
7921 ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct
7981 tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga
8041 tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat
8101 caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt
8161 gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg
8221 gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat
8281 tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg
8341 atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag
8401 ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca
8461 gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt
8521 ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc
8581 tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag
8641 gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg
8701 gtgatattct cattttagcc atttattatt tccttcctct tttctacagt atttaaagat
8761 accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa
8821 ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt
8881 atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta
8941 caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc
9001 gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg
9061 ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc
9121 cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg
9181 cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta
9241 attcggggga tctggatttt agtactggat tttggtttta ggaattagaa attttattga
9301 tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc aacacatgag
9361 cgaaacccta taggaaccct aattccctta tctgggaact actcacacat tattatggag
9421 aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct ttgccctcgg
9481 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
9541 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
9601 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
9661 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg gcgatcctgc
9721 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
9781 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
9841 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
9901 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
9961 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
10021 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
10081 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatagcctc
10141 cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
10201 ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa tgtcaagcac
10261 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
10321 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
10381 agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc
10441 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat ctcattgccc
10501 cccgggatct gcgaaagctc gagagagata gatttgtaga gagagactgg tgatttcagc
10561 gtgtcctctc caaatgaaat gaacttcctt atatagagga aggtcttgcg aaggatagtg
10621 ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga
10681 cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg
10741 accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt
10801 gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg
10861 gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc
10921 ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt
10981 atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct
11041 cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc
11101 ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt
11161 gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg
11221 ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta
11281 gacgagagtg tcgtgctcca ccatgttggc aagctgctct agccaatacg caaaccgcct
11341 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
11401 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
11461 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
11521 acaggaaaca gctatgacca tgattacgaa ttcgagctcg gtacccgggg atcctctaga
11581 gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
11641 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
11701 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
11761 atgctagagc agcttgagct tggatcagat tgtcgtttcc cgccttcagt ttagcttcat
11821 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
11881 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
11941 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
12001 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
12061 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
12121 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
12181 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
12241 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
12301 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactcttga
12361 c
//
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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pCAMBIA1303植物表达质粒
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