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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：
pCAMBIA1301
- 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：pCAMBIA1301植物表达质粒
别称: pCAMBIA1301
质粒属性
质粒简介
This vector contains a fully functional gusA reporter construct for simple and sensitive analysis of gene function or presence in regenerated plants by GUS assay. The construct uses E.coli gusA with an intron (from the castor bean catalase gene) inside the coding sequence to ensure that expression of glucuronidase activity is derived from eukaryotic cells, not from expression by residual A.tumefaciens cells. This vector is suitable for insertion of other genes of interest containing their own promoter and terminator. Researchers can excise the gusA gene and insert their own gene of interest in its place or use these vectors to create fusions of gusA with their gene of interest. These vectors contain the pUC18 polylinker-lacZa.
质粒图谱
质粒序列
LOCUS Exported 11849 bp ds-DNA circular SYN 13-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11849)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11849
/organism="synthetic DNA construct"
/mol_type="other DNA"
intron 10..199
/note="cat1 intron"
/note="castor bean catalase intron, modified"
CDS 2024..2041
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2076..2328
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2350..2374
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 3674..4303
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4737..5804
/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 5870..6064
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6408..6548
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6734..7322)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7409..8203)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8628..8652
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8730..8904
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8944..9969)
/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(10037..10714)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 10905..10926
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10941..10971
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 10979..10995
/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 11003..11019
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 11015..11248
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 11029..11085
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(11089..11105)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 11482..11827
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS join(11844..9,200..2017)
/codon_start=1
/product="beta-glucuronidase"
/note="GUS"
/note="This version of the gusA gene has a 5' extension
with a catalase intron to ensure expression in plants but
not bacteria."
/translation="MVDLRNRRLVRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWES
ALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGK
VWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGK
KKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSV
ELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELEVTAKSQTESDIYPLRVGIRS
VAVKGQQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYP
YAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQA
IKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCD
AHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDT
LAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKK
GIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQ"
ORIGIN
1 gatctgaggg taaatttcta gtttttctcc ttcattttct tggttaggac ccttttctct
61 ttttattttt ttgagctttg atctttcttt aaactgatct attttttaat tgattggtta
121 tggtgtaaat attacatagc tttaactgat aatctgatta ctttatttcg tgtgtctatg
181 atgatgatga tagttacaga accgacgact cgtccgtcct gtagaaaccc caacccgtga
241 aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga
301 tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt
361 taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg
421 cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt
481 cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta
541 tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac
601 cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga
661 cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca
721 tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac
781 gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga
841 tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag
901 cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga
961 actcgaagtc acagccaaaa gccagacaga gtctgatatc tacccgcttc gcgtcggcat
1021 ccggtcagtg gcagtgaagg gccaacagtt cctgattaac cacaaaccgt tctactttac
1081 tggctttggt cgtcatgaag atgcggactt acgtggcaaa ggattcgata acgtgctgat
1141 ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta
1201 cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga
1261 aactgctgct gtcggctttc agctgtcttt aggcattggt ttcgaagcgg gcaacaagcc
1321 gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc
1381 gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc
1441 caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc
1501 aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc
1561 tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg
1621 gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc
1681 ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc
1741 cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga
1801 tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt
1861 cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt
1921 cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat
1981 gaacttcggt gaaaaaccgc agcagggagg caaacaagct agccaccacc accaccacca
2041 cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg ttcaaacatt tggcaataaa
2101 gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga
2161 attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt
2221 ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg
2281 caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattaaact
2341 atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa
2401 taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
2461 caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt
2521 gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc
2581 taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt
2641 agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc
2701 gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac
2761 caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc
2821 accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt
2881 gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc
2941 gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc
3001 accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt
3061 tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag
3121 tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac
3181 caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg
3241 taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc
3301 ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa
3361 gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag
3421 agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa
3481 ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg
3541 ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt
3601 aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac
3661 gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac
3721 gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt
3781 cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct
3841 aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg
3901 cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa
3961 ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc
4021 cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc
4081 ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct
4141 ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg
4201 cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga
4261 ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag
4321 aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag
4381 gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg
4441 ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc
4501 gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag
4561 tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga
4621 cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt
4681 tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc
4741 gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt
4801 tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat
4861 cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg
4921 cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc
4981 tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt
5041 cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag
5101 aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg
5161 cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg
5221 gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg
5281 gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca
5341 tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct
5401 tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg
5461 agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg
5521 ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac
5581 gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg
5641 gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg
5701 acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc
5761 gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc
5821 tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca
5881 cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa
5941 agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg
6001 atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg
6061 cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc
6121 tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa
6181 tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg
6241 accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac
6301 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
6361 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
6421 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
6481 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
6541 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
6601 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
6661 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
6721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
6781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
6841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
6901 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
6961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
7021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
7081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
7141 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
7201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
7261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
7321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
7381 aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa atataatatt
7441 ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt
7501 cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg
7561 ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt
7621 tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta
7681 aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat
7741 ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc
7801 tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat
7861 acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc
7921 catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct
7981 ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat
8041 aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca
8101 ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt
8161 cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc
8221 ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga actccaattc
8281 actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc
8341 aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc
8401 agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc
8461 aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc
8521 cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt
8581 ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat
8641 tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg
8701 tactgaatta acgccgaatt aattcggggg atctggattt tagtactgga ttttggtttt
8761 aggaattaga aattttattg atagaagtat tttacaaata caaatacata ctaagggttt
8821 cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt atctgggaac
8881 tactcacaca ttattatgga gaaactcgag cttgtcgatc gacagatccg gtcggcatct
8941 actctatttc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac
9001 ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac
9061 agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc
9121 gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc
9181 ccggagtcgt ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg
9241 ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc
9301 cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac
9361 attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca
9421 aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt
9481 ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta
9541 ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc
9601 agcgatcgca tccatagcct ccgcgaccgg ttgtagaaca gcgggcagtt cggtttcagg
9661 caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct
9721 aaactcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata
9781 aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg
9841 ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc
9901 ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg
9961 ctttttcata tctcattgcc ccccgggatc tgcgaaagct cgagagagat agatttgtag
10021 agagagactg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg
10081 aaggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg agatatcaca
10141 tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt
10201 gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc
10261 tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa
10321 gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa
10381 agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag
10441 agtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg tggttggaac
10501 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc
10561 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc
10621 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag
10681 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt
10741 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttgg caagctgctc
10801 tagccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
10861 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
10921 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
10981 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc
11041 ggtacccggg gatcctctag agtcgacctg caggcatgca agcttggcac tggccgtcgt
11101 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
11161 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
11221 gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc
11281 ccgccttcag tttagcttca tggagtcaaa gattcaaata gaggacctaa cagaactcgc
11341 cgtaaagact ggcgaacagt tcatacagag tctcttacga ctcaatgaca agaagaaaat
11401 cttcgtcaac atggtggagc acgacacact tgtctactcc aaaaatatca aagatacagt
11461 ctcagaagac caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct
11521 cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg
11581 ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga
11641 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
11701 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
11761 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
11821 gagaacacgg gggactcttg accatggta
//
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：pCAMBIA1301植物表达质粒
别称: pCAMBIA1301
| 启动子: | 无，需要克隆进启动子才能表达 |
|---|---|
| 复制子: | pVS1 |
| 终止子: | 无 |
| 质粒大小: | 11849bp |
| 原核抗性: | Kan |
| 筛选标记: | Hyg,Gus |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 植物细胞 |
| 5'测序引物: | M13F |
| 3'测序引物: | M13R |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Kan |
| 筛选标记： | Hyg |
| 荧光蛋白： | Gus |
质粒简介
This vector contains a fully functional gusA reporter construct for simple and sensitive analysis of gene function or presence in regenerated plants by GUS assay. The construct uses E.coli gusA with an intron (from the castor bean catalase gene) inside the coding sequence to ensure that expression of glucuronidase activity is derived from eukaryotic cells, not from expression by residual A.tumefaciens cells. This vector is suitable for insertion of other genes of interest containing their own promoter and terminator. Researchers can excise the gusA gene and insert their own gene of interest in its place or use these vectors to create fusions of gusA with their gene of interest. These vectors contain the pUC18 polylinker-lacZa.
质粒图谱
质粒序列
LOCUS Exported 11849 bp ds-DNA circular SYN 13-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11849)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11849
/organism="synthetic DNA construct"
/mol_type="other DNA"
intron 10..199
/note="cat1 intron"
/note="castor bean catalase intron, modified"
CDS 2024..2041
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2076..2328
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2350..2374
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 3674..4303
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4737..5804
/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 5870..6064
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6408..6548
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6734..7322)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7409..8203)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8628..8652
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8730..8904
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8944..9969)
/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(10037..10714)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 10905..10926
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10941..10971
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 10979..10995
/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 11003..11019
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 11015..11248
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 11029..11085
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(11089..11105)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 11482..11827
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS join(11844..9,200..2017)
/codon_start=1
/product="beta-glucuronidase"
/note="GUS"
/note="This version of the gusA gene has a 5' extension
with a catalase intron to ensure expression in plants but
not bacteria."
/translation="MVDLRNRRLVRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWES
ALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGK
VWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGK
KKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSV
ELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELEVTAKSQTESDIYPLRVGIRS
VAVKGQQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYP
YAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQA
IKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCD
AHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDT
LAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKK
GIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQ"
ORIGIN
1 gatctgaggg taaatttcta gtttttctcc ttcattttct tggttaggac ccttttctct
61 ttttattttt ttgagctttg atctttcttt aaactgatct attttttaat tgattggtta
121 tggtgtaaat attacatagc tttaactgat aatctgatta ctttatttcg tgtgtctatg
181 atgatgatga tagttacaga accgacgact cgtccgtcct gtagaaaccc caacccgtga
241 aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga
301 tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt
361 taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg
421 cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt
481 cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta
541 tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac
601 cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga
661 cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca
721 tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac
781 gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga
841 tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag
901 cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga
961 actcgaagtc acagccaaaa gccagacaga gtctgatatc tacccgcttc gcgtcggcat
1021 ccggtcagtg gcagtgaagg gccaacagtt cctgattaac cacaaaccgt tctactttac
1081 tggctttggt cgtcatgaag atgcggactt acgtggcaaa ggattcgata acgtgctgat
1141 ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta
1201 cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga
1261 aactgctgct gtcggctttc agctgtcttt aggcattggt ttcgaagcgg gcaacaagcc
1321 gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc
1381 gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc
1441 caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc
1501 aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc
1561 tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg
1621 gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc
1681 ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc
1741 cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga
1801 tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt
1861 cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt
1921 cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat
1981 gaacttcggt gaaaaaccgc agcagggagg caaacaagct agccaccacc accaccacca
2041 cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg ttcaaacatt tggcaataaa
2101 gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga
2161 attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt
2221 ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg
2281 caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattaaact
2341 atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa
2401 taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
2461 caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt
2521 gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc
2581 taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt
2641 agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc
2701 gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac
2761 caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc
2821 accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt
2881 gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc
2941 gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc
3001 accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt
3061 tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag
3121 tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac
3181 caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg
3241 taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc
3301 ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa
3361 gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag
3421 agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa
3481 ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg
3541 ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt
3601 aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac
3661 gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac
3721 gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt
3781 cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct
3841 aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg
3901 cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa
3961 ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc
4021 cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc
4081 ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct
4141 ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg
4201 cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga
4261 ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag
4321 aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag
4381 gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg
4441 ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc
4501 gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag
4561 tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga
4621 cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt
4681 tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc
4741 gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt
4801 tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat
4861 cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg
4921 cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc
4981 tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt
5041 cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag
5101 aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg
5161 cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg
5221 gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg
5281 gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca
5341 tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct
5401 tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg
5461 agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg
5521 ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac
5581 gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg
5641 gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg
5701 acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc
5761 gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc
5821 tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca
5881 cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa
5941 agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg
6001 atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg
6061 cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc
6121 tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa
6181 tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg
6241 accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac
6301 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
6361 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
6421 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
6481 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
6541 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
6601 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
6661 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
6721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
6781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
6841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
6901 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
6961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
7021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
7081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
7141 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
7201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
7261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
7321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
7381 aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa atataatatt
7441 ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt
7501 cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg
7561 ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt
7621 tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta
7681 aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat
7741 ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc
7801 tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat
7861 acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc
7921 catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct
7981 ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat
8041 aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca
8101 ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt
8161 cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc
8221 ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga actccaattc
8281 actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc
8341 aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc
8401 agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc
8461 aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc
8521 cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt
8581 ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat
8641 tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg
8701 tactgaatta acgccgaatt aattcggggg atctggattt tagtactgga ttttggtttt
8761 aggaattaga aattttattg atagaagtat tttacaaata caaatacata ctaagggttt
8821 cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt atctgggaac
8881 tactcacaca ttattatgga gaaactcgag cttgtcgatc gacagatccg gtcggcatct
8941 actctatttc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac
9001 ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac
9061 agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc
9121 gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc
9181 ccggagtcgt ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg
9241 ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc
9301 cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac
9361 attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca
9421 aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt
9481 ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta
9541 ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc
9601 agcgatcgca tccatagcct ccgcgaccgg ttgtagaaca gcgggcagtt cggtttcagg
9661 caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct
9721 aaactcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata
9781 aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg
9841 ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc
9901 ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg
9961 ctttttcata tctcattgcc ccccgggatc tgcgaaagct cgagagagat agatttgtag
10021 agagagactg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg
10081 aaggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg agatatcaca
10141 tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt
10201 gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc
10261 tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa
10321 gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa
10381 agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag
10441 agtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg tggttggaac
10501 gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc
10561 agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc
10621 ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag
10681 gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt
10741 atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttgg caagctgctc
10801 tagccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
10861 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
10921 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
10981 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc
11041 ggtacccggg gatcctctag agtcgacctg caggcatgca agcttggcac tggccgtcgt
11101 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
11161 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
11221 gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc
11281 ccgccttcag tttagcttca tggagtcaaa gattcaaata gaggacctaa cagaactcgc
11341 cgtaaagact ggcgaacagt tcatacagag tctcttacga ctcaatgaca agaagaaaat
11401 cttcgtcaac atggtggagc acgacacact tgtctactcc aaaaatatca aagatacagt
11461 ctcagaagac caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct
11521 cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg
11581 ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga
11641 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
11701 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
11761 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
11821 gagaacacgg gggactcttg accatggta
//
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
| P2567/pENTR223-CYP19A1人源基因质粒 |
| P2568/pENTR223-SLC31A1人源基因质粒 |
| P2569/pENTR223-POP4人源基因质粒 |
| P2570/pENTR223-GGCT人源基因质粒 |
| P2572/pENTR223-PPIA人源基因质粒 |
| P2573/pENTR223-TRGV7人源基因质粒 |
| P2575/pENTR223-ZNF747人源基因质粒 |
| P2576/pENTR223-CHIC2人源基因质粒 |
| P2577/pENTR223-PSMD10人源基因质粒 |
| P2578/pENTR223-MRPL11人源基因质粒 |
| P2579/pENTR223-CHMP5人源基因质粒 |
| P2580/pENTR223-KDELR2-T11人源基因质粒 |
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