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- 详细信息
- 询价记录
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pCAMBIA1300-mCherry
- 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：pCAMBIA1300-mCherry植物荧光质粒
别称: pCAMBIA1300-mCherry
质粒属性
质粒简介
pCAMBIA1300-mCherry植物表达质粒
质粒图谱
质粒序列
LOCUS Exported 11182 bp ds-DNA circular SYN 06-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pCAMBIA1300-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11182)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Friday, July 6, 2018 from www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11182
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 220..244
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 1544..2173
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 2607..3674
/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 3740..3934
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 4278..4418
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(4604..5192)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5279..6073)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 6498..6522
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 6600..6774
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(6814..7839)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMD-AIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(7907..8584)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
promoter 9398..9743
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 9752..9801
/note="MCS"
CDS 9804..10514
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/note="mCherry"
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
terminator 10534..11175
/note="E9 terminator"
/note="terminator and polyadenylation signal from the pea
rbcS-E9 gene"
ORIGIN
1 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
61 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
121 cccttcccaa cagttgcgca gcctgaatgg cgaatgctag agcagcttga gcttggatca
181 gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt
241 aaacctaaga gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt
301 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt
361 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg
421 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
481 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
541 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
601 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
661 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
721 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
781 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
841 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
901 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
961 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
1021 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
1081 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
1141 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
1201 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
1261 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt
1321 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt
1381 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
1441 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
1501 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
1561 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
1621 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
1681 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
1741 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
1801 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
1861 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
1921 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
1981 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
2041 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
2101 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
2161 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
2221 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
2281 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
2341 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
2401 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
2461 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
2521 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac
2581 ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg
2641 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac
2701 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca
2761 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg
2821 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca
2881 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga
2941 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca
3001 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc
3061 gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg
3121 tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct
3181 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc
3241 gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg
3301 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca
3361 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg
3421 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat
3481 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt
3541 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg
3601 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat
3661 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag
3721 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg
3781 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt
3841 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta
3901 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag
3961 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc
4021 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag
4081 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc
4141 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
4201 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
4261 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
4321 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
4381 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
5041 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact
5281 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca
5341 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga
5401 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc
5461 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga
5521 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa
5581 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt
5641 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga
5701 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt
5761 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc
5821 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata
5881 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca
5941 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc
6001 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc
6061 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga
6121 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata
6181 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga
6241 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
6301 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
6361 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
6421 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
6481 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
6541 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg
6601 atctggattt tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat
6661 tttacaaata caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct
6721 ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag
6781 cttgtcgatc gacagatccg gtcggcatct actctatttc tttgccctcg gacgagtgct
6841 ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc
6901 gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc
6961 gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag
7021 ttggtcaaga ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg
7081 atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa
7141 gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac
7201 cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag
7261 gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac
7321 ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat
7381 cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc
7441 gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg
7501 ttgtagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg
7561 gcgggagatg caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat
7621 cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc
7681 gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga
7741 ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa
7801 cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccgggatc
7861 tgcgaaagct cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct
7921 ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt gggattgtgc
7981 gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg
8041 aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc
8101 ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc
8161 accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag
8221 gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac
8281 tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tatcacatca
8341 atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg
8401 tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt
8461 atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg
8521 acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt
8581 ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt
8641 gtcgtgctcc accatgttgg caagctgctc tagccaatac gcaaaccgcc tctccccgcg
8701 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
8761 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
8821 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
8881 agctatgaca tgattacgaa ttcggtcccc agattagcct tttcaatttc agaaagaatg
8941 ctaacccaca gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga
9001 gcaataatct ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat
9061 tcaggactaa ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta
9121 ttccagtatg gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag
9181 tctctaaaaa ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg
9241 acctaacaga actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca
9301 atgacaagaa gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa
9361 atatcaaaga tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa
9421 tatccggaaa cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag
9481 tggaaaagga aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg
9541 aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg
9601 aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg
9661 acgtaaggga tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa
9721 gttcatttca tttggagaga acacggggga cgagctcaag cttcgaattc tgcagtcgac
9781 ggtaccgcgg gcccgggatc ctgatggtga gcaagggcga ggaggataac atggccatca
9841 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
9901 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
9961 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
10021 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
10081 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
10141 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
10201 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
10261 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
10321 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
10381 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
10441 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
10501 agctgtacaa gtgatctagg ggctagaatg cagagctttc gttcgtatca tcggtttcga
10561 caacgttcgt caagttcaat gcatcagttt cattgcgcac acaccagaat cctactgagt
10621 ttgagtatta tggcattggg aaaactgttt ttcttgtacc atttgttgtg cttgtaattt
10681 actgtgtttt ttattcggtt ttcgctatcg aactgtgaaa tggaaatgga tggagaagag
10741 ttaatgaatg atatggtcct tttgttcatt ctcaaattaa tattatttgt tttttctctt
10801 atttgttgtg tgttgaattt gaaattataa gagatatgca aacattttgt tttgagtaaa
10861 aatgtgtcaa atcgtggcct ctaatgaccg aagttaatat gaggagtaaa acacttgtag
10921 ttgtaccatt atgcttattc actaggcaac aaatatattt tcagacctag aaaagctgca
10981 aatgttactg aatacaagta tgtcctcttg tgttttagac atttatgaac tttcctttat
11041 gtaattttcc agaatccttg tcagattcta atcattgctt tataattata gttatactca
11101 tggatttgta gttgagtatg aaaatatttt ttaatgcatt ttatgacttg ccaattgatt
11161 gacaacatgc atcaagcttg gc
//
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：pCAMBIA1300-mCherry植物荧光质粒
别称: pCAMBIA1300-mCherry
| 启动子: | 35s |
|---|---|
| 复制子: | pVS1 |
| 终止子: | E9 terminator |
| 质粒分类: | 植物红色荧光表达质粒 |
| 质粒大小: | 11182bp |
| 质粒标签: | 红色荧光蛋白C-mCherry |
| 原核抗性: | Kan |
| 筛选标记: | Hyg |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 5'测序引物: | 根据序列设计引物 |
| 3'测序引物: | 根据序列设计引物 |
| 备注: | 农杆菌介导 |
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： | 红色 |
质粒简介
pCAMBIA1300-mCherry植物表达质粒
质粒图谱
质粒序列
LOCUS Exported 11182 bp ds-DNA circular SYN 06-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pCAMBIA1300-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11182)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Friday, July 6, 2018 from www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11182
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 220..244
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 1544..2173
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 2607..3674
/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 3740..3934
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 4278..4418
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(4604..5192)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5279..6073)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 6498..6522
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 6600..6774
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(6814..7839)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMD-AIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(7907..8584)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
promoter 9398..9743
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 9752..9801
/note="MCS"
CDS 9804..10514
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/note="mCherry"
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
terminator 10534..11175
/note="E9 terminator"
/note="terminator and polyadenylation signal from the pea
rbcS-E9 gene"
ORIGIN
1 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
61 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
121 cccttcccaa cagttgcgca gcctgaatgg cgaatgctag agcagcttga gcttggatca
181 gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt
241 aaacctaaga gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt
301 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt
361 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg
421 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
481 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
541 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
601 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
661 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
721 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
781 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
841 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
901 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
961 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
1021 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
1081 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
1141 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
1201 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
1261 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt
1321 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt
1381 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
1441 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
1501 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
1561 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
1621 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
1681 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
1741 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
1801 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
1861 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
1921 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
1981 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
2041 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
2101 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
2161 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
2221 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
2281 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
2341 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
2401 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
2461 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
2521 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac
2581 ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg
2641 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac
2701 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca
2761 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg
2821 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca
2881 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga
2941 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca
3001 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc
3061 gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg
3121 tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct
3181 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc
3241 gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg
3301 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca
3361 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg
3421 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat
3481 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt
3541 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg
3601 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat
3661 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag
3721 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg
3781 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt
3841 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta
3901 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag
3961 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc
4021 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag
4081 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc
4141 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
4201 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
4261 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
4321 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
4381 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
5041 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact
5281 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca
5341 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga
5401 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc
5461 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga
5521 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa
5581 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt
5641 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga
5701 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt
5761 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc
5821 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata
5881 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca
5941 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc
6001 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc
6061 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga
6121 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata
6181 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga
6241 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
6301 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
6361 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
6421 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
6481 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
6541 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg
6601 atctggattt tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat
6661 tttacaaata caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct
6721 ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag
6781 cttgtcgatc gacagatccg gtcggcatct actctatttc tttgccctcg gacgagtgct
6841 ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc
6901 gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc
6961 gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag
7021 ttggtcaaga ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg
7081 atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa
7141 gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac
7201 cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag
7261 gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac
7321 ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat
7381 cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc
7441 gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg
7501 ttgtagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg
7561 gcgggagatg caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat
7621 cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc
7681 gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga
7741 ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa
7801 cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccgggatc
7861 tgcgaaagct cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct
7921 ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt gggattgtgc
7981 gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg
8041 aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc
8101 ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc
8161 accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag
8221 gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac
8281 tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tatcacatca
8341 atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg
8401 tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt
8461 atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg
8521 acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt
8581 ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt
8641 gtcgtgctcc accatgttgg caagctgctc tagccaatac gcaaaccgcc tctccccgcg
8701 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
8761 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
8821 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
8881 agctatgaca tgattacgaa ttcggtcccc agattagcct tttcaatttc agaaagaatg
8941 ctaacccaca gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga
9001 gcaataatct ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat
9061 tcaggactaa ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta
9121 ttccagtatg gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag
9181 tctctaaaaa ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg
9241 acctaacaga actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca
9301 atgacaagaa gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa
9361 atatcaaaga tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa
9421 tatccggaaa cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag
9481 tggaaaagga aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg
9541 aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg
9601 aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg
9661 acgtaaggga tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa
9721 gttcatttca tttggagaga acacggggga cgagctcaag cttcgaattc tgcagtcgac
9781 ggtaccgcgg gcccgggatc ctgatggtga gcaagggcga ggaggataac atggccatca
9841 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
9901 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
9961 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
10021 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
10081 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
10141 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
10201 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
10261 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
10321 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
10381 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
10441 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
10501 agctgtacaa gtgatctagg ggctagaatg cagagctttc gttcgtatca tcggtttcga
10561 caacgttcgt caagttcaat gcatcagttt cattgcgcac acaccagaat cctactgagt
10621 ttgagtatta tggcattggg aaaactgttt ttcttgtacc atttgttgtg cttgtaattt
10681 actgtgtttt ttattcggtt ttcgctatcg aactgtgaaa tggaaatgga tggagaagag
10741 ttaatgaatg atatggtcct tttgttcatt ctcaaattaa tattatttgt tttttctctt
10801 atttgttgtg tgttgaattt gaaattataa gagatatgca aacattttgt tttgagtaaa
10861 aatgtgtcaa atcgtggcct ctaatgaccg aagttaatat gaggagtaaa acacttgtag
10921 ttgtaccatt atgcttattc actaggcaac aaatatattt tcagacctag aaaagctgca
10981 aatgttactg aatacaagta tgtcctcttg tgttttagac atttatgaac tttcctttat
11041 gtaattttcc agaatccttg tcagattcta atcattgctt tataattata gttatactca
11101 tggatttgta gttgagtatg aaaatatttt ttaatgcatt ttatgacttg ccaattgatt
11161 gacaacatgc atcaagcttg gc
//
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
| P3836/pCP20质粒菌种 |
| P2837/pREDKI大肠敲除质粒 |
| P4937/pGRG36大肠敲除质粒 |
| P4945/pGRG37大肠敲除质粒 |
| P0762/pCas大肠敲除质粒 |
| P0116/pCas9大肠敲除质粒 |
| P4855/pCRISPomyces-1大肠敲除质粒 |
| P1031/pCRISPomyces-2大肠敲除质粒 |
| P1931/BPK764大肠敲除质粒 |
| P1932/MSP1673大肠敲除质粒 |
| P1827/pJYS3_ΔcrtYf大肠敲除质粒 |
| P0748/pET-NLS-Cas9-6xHis大肠敲除质粒 |
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