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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：
pCXGFP-P-pXcmI-GFP
- 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：pCXGFP-P-pXcmI-GFP植物表达质粒
别称: pCXGFP-P-pXcmI-GFP
质粒属性
质粒简介
pCXGFP-P质粒是一个植物启动子荧光检测载体，通过GFP的荧光强度来检测启动子活性高低。使用XcmI酶切空载体得到T载体，可连接带A的PCR产物，并在ccdb致死基因的帮助下，基因克隆的阳性率得以大大提高。
质粒图谱
质粒序列
LOCUS Exported 10640 bp ds-DNA circular SYN 28-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10640)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..10640
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 9..261
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(278..994)
/codon_start=1
/label=GFP
/translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTMGYGVLCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
CDS 1345..1650
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
misc_feature 1964..1988
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 3287..3916
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4350..5417
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 5483..5677
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6021..6161
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(6347..6935)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7022..7816)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8241..8265
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8343..8517
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8557..9582)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(9649..10326)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
ORIGIN
1 gaattcccga tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg
61 ctatattttg ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaac
121 ccatctcata aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaa
181 cagaaattat atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaacttta
241 ttgccaaatg tttgaacgat cggggaaatt cgagctctca tttgtatagt tcatccatgc
301 catgtgtaat cccagcagct gttacaaact caagaaggac catgtggtct ctcttttcgt
361 tgggatcttt cgaaagggca gattgtgtgg acaggtaatg gttgtctggt aaaaggacag
421 ggccatcgcc aattggagta ttttgttgat aatggtctgc tagttgaacg cttccatctt
481 caatgttgtg tctaattttg aagttaactt tgattccatt cttttgtttg tctgccatga
541 tgtatacatt gtgtgagtta tagttgtatt ccaatttgtg tccaagaatg tttccatctt
601 ctttaaaatc aatacctttt aactcgattc tattaacaag ggtatcacct tcaaacttga
661 cttcagcacg tgtcttgtag ttcccgtcat ctttgaaaaa tatagttctt tcctgtacat
721 aaccttcggg catggcactc ttgaaaaagt catgccgttt catatgatct gggtatcttg
781 aaaagcatag aacaccataa cccatagtag tgacaagtgt tggccatgga acaggtagtt
841 ttccagtagt gcaaataaat ttaagggtaa gttttccgta tgttgcatca ccttcaccct
901 ctccactgac agaaaatttg tgcccattaa catcaccatc taattcaaca agaattggga
961 caactccagt gaaaagttct tctcctttac ccatggatcc ccaatacttg tatggggctt
1021 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
1081 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
1141 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
1201 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
1261 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
1321 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
1381 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
1441 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
1501 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
1561 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
1621 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct
1681 gcaggtcgac ccatacaagt attggggatc ctctagagtc gacctgcagg catgcaagct
1741 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
1801 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
1861 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ctagagcagc ttgagcttgg
1921 atcagattgt cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggc
1981 gggtaaacct aagagaaaag agcgtttatt agaataacgg atatttaaaa gggcgtgaaa
2041 aggtttatcc gttcgtccat ttgtatgtgc atgccaacca cagggttccc ctcgggatca
2101 aagtactttg atccaacccc tccgctgcta tagtgcagtc ggcttctgac gttcagtgca
2161 gccgtcttct gaaaacgaca tgtcgcacaa gtcctaagtt acgcgacagg ctgccgccct
2221 gcccttttcc tggcgttttc ttgtcgcgtg ttttagtcgc ataaagtaga atacttgcga
2281 ctagaaccgg agacattacg ccatgaacaa gagcgccgcc gctggcctgc tgggctatgc
2341 ccgcgtcagc accgacgacc aggacttgac caaccaacgg gccgaactgc acgcggccgg
2401 ctgcaccaag ctgttttccg agaagatcac cggcaccagg cgcgaccgcc cggagctggc
2461 caggatgctt gaccacctag ccctggcgac gttgtgacag tgaccaggct agaccgcctg
2521 gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc
2581 ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg
2641 accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc
2701 gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg
2761 gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg
2821 gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa
2881 gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc
2941 gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg
3001 acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg
3061 ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg
3121 gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc
3181 gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg
3241 cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc
3301 tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc
3361 cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg
3421 cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac
3481 gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc
3541 ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt
3601 gcagccaagc ccttacgaca tatgggcaac cgccgacctg gtggagctgg ttaagcagcg
3661 cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg
3721 cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga
3781 gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct
3841 tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa
3901 atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta
3961 agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac
4021 acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag
4081 atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag
4141 ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg
4201 cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg
4261 aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg
4321 aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg
4381 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc
4441 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc
4501 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg
4561 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc
4621 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg
4681 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg
4741 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga
4801 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg
4861 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa
4921 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg
4981 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga
5041 gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga
5101 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc
5161 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca
5221 gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct
5281 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg
5341 aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag
5401 catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa
5461 aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca
5521 ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca
5581 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac
5641 ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg
5701 aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc
5761 gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac
5821 cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc
5881 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
5941 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
6001 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
6061 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
6121 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
6181 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
6241 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
6301 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
6361 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
6421 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
6481 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
6541 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
6601 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
6661 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
6721 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
6781 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
6841 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
6901 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
6961 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg cattctaggt
7021 actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc
7081 ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgatcgacc
7141 ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa
7201 agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa
7261 agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt
7321 taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta
7381 agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt
7441 cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt
7501 gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc
7561 tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc
7621 atagcatcat gtccttttcc cgttccacat cataggttgt ccctttatac cggctgtccg
7681 tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca
7741 ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata
7801 ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa agatacccca
7861 agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa
7921 ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac
7981 ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca
8041 acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt
8101 ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg
8161 ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg
8221 gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga
8281 caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg
8341 gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag
8401 tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac
8461 cctataggaa ccctaattcc cttatctggg aactactcac acattattat ggagaaactc
8521 gagcttgtcg atcgacagat ccggtcggca tctactctat ttctttgccc tcggacgagt
8581 gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc
8641 cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc
8701 gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata
8761 gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc ctgcaagctc
8821 cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca
8881 gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat
8941 gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac
9001 gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc
9061 gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc
9121 aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg
9181 gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag cctccgcgac
9241 cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc
9301 acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa gcacttccgg
9361 aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc
9421 ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg
9481 agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag
9541 aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt gccccccgga
9601 tctgcgaaag ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct
9661 ctccaaatga aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt
9721 gcgtcatccc ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt
9781 ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg
9841 tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg
9901 ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg
9961 aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag
10021 actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat
10081 caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg
10141 ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct
10201 ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag
10261 tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa
10321 gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga
10381 gtgtcgtgct ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg
10441 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
10501 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
10561 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
10621 acagctatga ccatgattac
//
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：pCXGFP-P-pXcmI-GFP植物表达质粒
别称: pCXGFP-P-pXcmI-GFP
| 质粒大小: | 10640bp |
|---|---|
| 原核抗性: | Kan |
| 筛选标记: | Hyg |
| 克隆菌株: | DB3.1 |
| 培养条件: | 37度 |
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： |
pCXGFP-P质粒是一个植物启动子荧光检测载体，通过GFP的荧光强度来检测启动子活性高低。使用XcmI酶切空载体得到T载体，可连接带A的PCR产物，并在ccdb致死基因的帮助下，基因克隆的阳性率得以大大提高。
质粒图谱
质粒序列
LOCUS Exported 10640 bp ds-DNA circular SYN 28-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10640)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..10640
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 9..261
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(278..994)
/codon_start=1
/label=GFP
/translation="MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTMGYGVLCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
CDS 1345..1650
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
misc_feature 1964..1988
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 3287..3916
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4350..5417
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 5483..5677
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6021..6161
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(6347..6935)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7022..7816)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8241..8265
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8343..8517
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8557..9582)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(9649..10326)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
ORIGIN
1 gaattcccga tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg
61 ctatattttg ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaac
121 ccatctcata aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaa
181 cagaaattat atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaacttta
241 ttgccaaatg tttgaacgat cggggaaatt cgagctctca tttgtatagt tcatccatgc
301 catgtgtaat cccagcagct gttacaaact caagaaggac catgtggtct ctcttttcgt
361 tgggatcttt cgaaagggca gattgtgtgg acaggtaatg gttgtctggt aaaaggacag
421 ggccatcgcc aattggagta ttttgttgat aatggtctgc tagttgaacg cttccatctt
481 caatgttgtg tctaattttg aagttaactt tgattccatt cttttgtttg tctgccatga
541 tgtatacatt gtgtgagtta tagttgtatt ccaatttgtg tccaagaatg tttccatctt
601 ctttaaaatc aatacctttt aactcgattc tattaacaag ggtatcacct tcaaacttga
661 cttcagcacg tgtcttgtag ttcccgtcat ctttgaaaaa tatagttctt tcctgtacat
721 aaccttcggg catggcactc ttgaaaaagt catgccgttt catatgatct gggtatcttg
781 aaaagcatag aacaccataa cccatagtag tgacaagtgt tggccatgga acaggtagtt
841 ttccagtagt gcaaataaat ttaagggtaa gttttccgta tgttgcatca ccttcaccct
901 ctccactgac agaaaatttg tgcccattaa catcaccatc taattcaaca agaattggga
961 caactccagt gaaaagttct tctcctttac ccatggatcc ccaatacttg tatggggctt
1021 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
1081 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
1141 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
1201 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
1261 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
1321 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
1381 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
1441 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
1501 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
1561 gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
1621 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct
1681 gcaggtcgac ccatacaagt attggggatc ctctagagtc gacctgcagg catgcaagct
1741 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
1801 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
1861 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ctagagcagc ttgagcttgg
1921 atcagattgt cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggc
1981 gggtaaacct aagagaaaag agcgtttatt agaataacgg atatttaaaa gggcgtgaaa
2041 aggtttatcc gttcgtccat ttgtatgtgc atgccaacca cagggttccc ctcgggatca
2101 aagtactttg atccaacccc tccgctgcta tagtgcagtc ggcttctgac gttcagtgca
2161 gccgtcttct gaaaacgaca tgtcgcacaa gtcctaagtt acgcgacagg ctgccgccct
2221 gcccttttcc tggcgttttc ttgtcgcgtg ttttagtcgc ataaagtaga atacttgcga
2281 ctagaaccgg agacattacg ccatgaacaa gagcgccgcc gctggcctgc tgggctatgc
2341 ccgcgtcagc accgacgacc aggacttgac caaccaacgg gccgaactgc acgcggccgg
2401 ctgcaccaag ctgttttccg agaagatcac cggcaccagg cgcgaccgcc cggagctggc
2461 caggatgctt gaccacctag ccctggcgac gttgtgacag tgaccaggct agaccgcctg
2521 gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc
2581 ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg
2641 accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc
2701 gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg
2761 gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg
2821 gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa
2881 gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc
2941 gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg
3001 acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg
3061 ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg
3121 gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc
3181 gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg
3241 cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc
3301 tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc
3361 cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg
3421 cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac
3481 gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc
3541 ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt
3601 gcagccaagc ccttacgaca tatgggcaac cgccgacctg gtggagctgg ttaagcagcg
3661 cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg
3721 cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga
3781 gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct
3841 tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa
3901 atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta
3961 agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac
4021 acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag
4081 atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag
4141 ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg
4201 cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg
4261 aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg
4321 aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg
4381 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc
4441 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc
4501 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg
4561 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc
4621 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg
4681 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg
4741 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga
4801 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg
4861 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa
4921 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg
4981 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga
5041 gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga
5101 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc
5161 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca
5221 gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct
5281 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg
5341 aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag
5401 catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa
5461 aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca
5521 ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca
5581 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac
5641 ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg
5701 aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc
5761 gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac
5821 cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc
5881 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
5941 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
6001 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
6061 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
6121 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
6181 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
6241 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
6301 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
6361 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
6421 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
6481 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
6541 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
6601 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
6661 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
6721 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
6781 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
6841 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
6901 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
6961 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg cattctaggt
7021 actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc
7081 ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgatcgacc
7141 ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa
7201 agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa
7261 agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt
7321 taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta
7381 agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt
7441 cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt
7501 gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc
7561 tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc
7621 atagcatcat gtccttttcc cgttccacat cataggttgt ccctttatac cggctgtccg
7681 tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca
7741 ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata
7801 ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa agatacccca
7861 agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa
7921 ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac
7981 ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca
8041 acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt
8101 ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg
8161 ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg
8221 gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga
8281 caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg
8341 gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag
8401 tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac
8461 cctataggaa ccctaattcc cttatctggg aactactcac acattattat ggagaaactc
8521 gagcttgtcg atcgacagat ccggtcggca tctactctat ttctttgccc tcggacgagt
8581 gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc
8641 cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc
8701 gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata
8761 gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc ctgcaagctc
8821 cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca
8881 gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat
8941 gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac
9001 gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc
9061 gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc
9121 aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg
9181 gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag cctccgcgac
9241 cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc
9301 acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa gcacttccgg
9361 aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc
9421 ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg
9481 agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag
9541 aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt gccccccgga
9601 tctgcgaaag ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct
9661 ctccaaatga aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt
9721 gcgtcatccc ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt
9781 ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg
9841 tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg
9901 ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg
9961 aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag
10021 actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat
10081 caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg
10141 ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct
10201 ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag
10261 tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa
10321 gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga
10381 gtgtcgtgct ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg
10441 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
10501 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
10561 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
10621 acagctatga ccatgattac
//
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
| P3324/pCMV-SPORT6-PSAT1（1同义突变）人源基因质粒 |
| P3325/pCMV-SPORT6-MGMT人源基因质粒 |
| P3326/pCMV-SPORT6-PPIH人源基因质粒 |
| P3327/pCMV-SPORT6-PREB人源基因质粒 |
| P3328/pCMV-SPORT6-PPP5C人源基因质粒 |
| P3329/pCMV-SPORT6-TMEM33人源基因质粒 |
| P3330/pCMV-SPORT6-TNFRSF10B（2点突变）人源基因质粒 |
| P3331/pCMV-SPORT6-RAD1人源基因质粒 |
| P3332/pCMV-SPORT6-PAPOLA人源基因质粒 |
| P3333/pCMV-SPORT6-ZNF160（1点突变）人源基因质粒 |
| P3334/pCMV-SPORT6-SLX1A人源基因质粒 |
| P3335/pCMV-SPORT6-LY6K人源基因质粒 |
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