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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pCAMBIA3301
- 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：pCAMBIA3301植物表达质粒
别称: pCAMBIA3301
质粒属性
质粒简介
pCAMBIA3301是一个植物细胞表达载体质粒。
质粒图谱
质粒序列
LOCUS Exported 11319 bp ds-DNA circular SYN 12-MAY-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11319)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11319
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS join(2..16,207..2024)
/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"
intron 17..206
/note="cat1 intron"
/note="castor bean catalase intron, modified"
CDS 2031..2048
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2083..2335
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2357..2381
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 3681..4310
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4744..5811
/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 5877..6071
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6415..6555
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6741..7329)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7416..8210)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8635..8659
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8737..8911
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8918..9469)
/codon_start=1
/gene="Streptomyces hygroscopicus bar"
/product="phosphinothricin acetyltransferase"
/note="BlpR"
/note="confers resistance to bialophos or phosphinothricin"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
promoter complement(9514..10191)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 10382..10403
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10418..10448
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 10456..10472
/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 10480..10496
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 10492..10725
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 10506..10562
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(10566..10582)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 10959..11304
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
ORIGIN
1 catggtagat ctgagggtaa atttctagtt tttctccttc attttcttgg ttaggaccct
61 tttctctttt tatttttttg agctttgatc tttctttaaa ctgatctatt ttttaattga
121 ttggttatgg tgtaaatatt acatagcttt aactgataat ctgattactt tatttcgtgt
181 gtctatgatg atgatgatag ttacagaacc gacgactcgt ccgtcctgta gaaaccccaa
241 cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg
301 gaattgatca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag
361 gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgcgggc aacgtctggt
421 atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg
481 atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg
541 gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac
601 gtatcaccgt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga
661 ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactatgccg
721 gaatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg
781 tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca
841 atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag
901 gcactagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc
961 tctatgaact cgaagtcaca gccaaaagcc agacagagtc tgatatctac ccgcttcgcg
1021 tcggcatccg gtcagtggca gtgaagggcc aacagttcct gattaaccac aaaccgttct
1081 actttactgg ctttggtcgt catgaagatg cggacttacg tggcaaagga ttcgataacg
1141 tgctgatggt gcacgaccac gcattaatgg actggattgg ggccaactcc taccgtacct
1201 cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga
1261 ttgatgaaac tgctgctgtc ggctttcagc tgtctttagg cattggtttc gaagcgggca
1321 acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caagcgcact
1381 tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga
1441 gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg
1501 cggaagcaac gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct
1561 gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt
1621 acggatggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac
1681 ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata
1741 cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat
1801 ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat
1861 ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag
1921 ggatcttcac tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga
1981 ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaagctagc caccaccacc
2041 accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg
2101 caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt
2161 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga
2221 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata
2281 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa
2341 ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt
2401 attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg
2461 tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg
2521 ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca
2581 caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc
2641 gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa
2701 caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt
2761 gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat
2821 caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg
2881 cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga
2941 cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc
3001 cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt
3061 cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg
3121 cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct
3181 gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc
3241 gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg
3301 cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga
3361 acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt
3421 accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac
3481 gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc
3541 tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta
3601 tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa
3661 caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag
3721 gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc
3781 tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc
3841 aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg
3901 gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg
3961 cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg
4021 ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac
4081 aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg
4141 aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct
4201 acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg
4261 cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg
4321 taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag
4381 cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt
4441 tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac
4501 cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat
4561 aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag
4621 gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg
4681 gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt
4741 gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg
4801 gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc
4861 ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca
4921 gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt
4981 ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc
5041 cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg
5101 cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta
5161 ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac
5221 aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc
5281 gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac
5341 gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt
5401 gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc
5461 gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg
5521 ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc
5581 ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa
5641 cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg
5701 tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta
5761 gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag
5821 cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg
5881 gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg
5941 aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa
6001 aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg
6061 gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt
6121 cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc
6181 tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg
6241 ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg
6301 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
6361 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
6421 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
6481 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
6541 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
6601 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
6661 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
6721 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
6781 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
6841 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
6901 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
6961 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
7021 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
7081 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
7141 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
7201 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
7261 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
7321 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
7381 tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata
7441 taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca
7501 tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac
7561 ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca
7621 aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc
7681 gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt
7741 tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg
7801 tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac
7861 tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa
7921 aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc
7981 aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc
8041 acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt
8101 tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg
8161 tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc
8221 cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact
8281 ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt
8341 tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat
8401 cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc
8461 gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa
8521 agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt
8581 atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag
8641 gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt
8701 tttaatgtac tgaattaacg ccgaattaat tcgggggatc tggattttag tactggattt
8761 tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa atacatacta
8821 agggtttctt atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatc
8881 tgggaactac tcacacatta ttatggagaa actcgagtca aatctcggtg acgggcagga
8941 ccggacgggg cggtaccggc aggctgaagt ccagctgcca gaaacccacg tcatgccagt
9001 tcccgtgctt gaagccggcc gcccgcagca tgccgcgggg ggcatatccg agcgcctcgt
9061 gcatgcgcac gctcgggtcg ttgggcagcc cgatgacagc gaccacgctc ttgaagccct
9121 gtgcctccag ggacttcagc aggtgggtgt agagcgtgga gcccagtccc gtccgctggt
9181 ggcgggggga gacgtacacg gtcgactcgg ccgtccagtc gtaggcgttg cgtgccttcc
9241 aggggcccgc gtaggcgatg ccggcgacct cgccgtccac ctcggcgacg agccagggat
9301 agcgctcccg cagacggacg aggtcgtccg tccactcctg cggttcctgc ggctcggtac
9361 ggaagttgac cgtgcttgtc tcgatgtagt ggttgacgat ggtgcagacc gccggcatgt
9421 ccgcctcggt ggcacggcgg atgtcggccg ggcgtcgttc tgggctcatg gtagactcga
9481 gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca aatgaaatga
9541 acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg
9601 tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt
9661 tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct
9721 tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc ttccttttct
9781 actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag gtttcccgat
9841 attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgata
9901 ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc cacttgcttt
9961 gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct
10021 ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc gcaatgatgg
10081 catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca gatagctggg
10141 caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc aatagccctt
10201 tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc gtgctccacc
10261 atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
10321 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
10381 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
10441 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
10501 attacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc
10561 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
10621 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
10681 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag cttgagcttg
10741 gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat tcaaatagag
10801 gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct cttacgactc
10861 aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt ctactccaaa
10921 aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta
10981 atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata
11041 gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt
11101 gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg
11161 gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact
11221 gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga
11281 agttcatttc atttggagag aacacggggg actcttgac
//
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：pCAMBIA3301植物表达质粒
别称: pCAMBIA3301
| 启动子: | 无，需要克隆启动子才能表达 |
|---|---|
| 复制子: | pVS1 |
| 终止子: | 无 |
| 质粒分类: | 植物系列，蛋白过表达载体 |
| 质粒大小: | 11319bp |
| 原核抗性: | Kan |
| 筛选标记: | Bar |
| 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:TGTAAAACGACGGCCAGT |
| 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： | Bar |
| 荧光蛋白： |
pCAMBIA3301是一个植物细胞表达载体质粒。
质粒图谱
质粒序列
LOCUS Exported 11319 bp ds-DNA circular SYN 12-MAY-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11319)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..11319
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS join(2..16,207..2024)
/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"
intron 17..206
/note="cat1 intron"
/note="castor bean catalase intron, modified"
CDS 2031..2048
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2083..2335
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2357..2381
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 3681..4310
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 4744..5811
/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 5877..6071
/note="pVS1 oriV"
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 6415..6555
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6741..7329)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7416..8210)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8635..8659
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8737..8911
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8918..9469)
/codon_start=1
/gene="Streptomyces hygroscopicus bar"
/product="phosphinothricin acetyltransferase"
/note="BlpR"
/note="confers resistance to bialophos or phosphinothricin"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
promoter complement(9514..10191)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 10382..10403
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10418..10448
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 10456..10472
/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 10480..10496
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 10492..10725
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
misc_feature 10506..10562
/note="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(10566..10582)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 10959..11304
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
ORIGIN
1 catggtagat ctgagggtaa atttctagtt tttctccttc attttcttgg ttaggaccct
61 tttctctttt tatttttttg agctttgatc tttctttaaa ctgatctatt ttttaattga
121 ttggttatgg tgtaaatatt acatagcttt aactgataat ctgattactt tatttcgtgt
181 gtctatgatg atgatgatag ttacagaacc gacgactcgt ccgtcctgta gaaaccccaa
241 cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg
301 gaattgatca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag
361 gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgcgggc aacgtctggt
421 atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg
481 atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg
541 gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac
601 gtatcaccgt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga
661 ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactatgccg
721 gaatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg
781 tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca
841 atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag
901 gcactagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc
961 tctatgaact cgaagtcaca gccaaaagcc agacagagtc tgatatctac ccgcttcgcg
1021 tcggcatccg gtcagtggca gtgaagggcc aacagttcct gattaaccac aaaccgttct
1081 actttactgg ctttggtcgt catgaagatg cggacttacg tggcaaagga ttcgataacg
1141 tgctgatggt gcacgaccac gcattaatgg actggattgg ggccaactcc taccgtacct
1201 cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga
1261 ttgatgaaac tgctgctgtc ggctttcagc tgtctttagg cattggtttc gaagcgggca
1321 acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caagcgcact
1381 tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga
1441 gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg
1501 cggaagcaac gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct
1561 gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt
1621 acggatggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac
1681 ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata
1741 cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat
1801 ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat
1861 ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag
1921 ggatcttcac tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga
1981 ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaagctagc caccaccacc
2041 accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg
2101 caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt
2161 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga
2221 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata
2281 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa
2341 ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt
2401 attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg
2461 tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg
2521 ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca
2581 caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc
2641 gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa
2701 caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt
2761 gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat
2821 caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg
2881 cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga
2941 cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc
3001 cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt
3061 cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg
3121 cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct
3181 gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc
3241 gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg
3301 cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga
3361 acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt
3421 accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac
3481 gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc
3541 tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta
3601 tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa
3661 caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag
3721 gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc
3781 tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc
3841 aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg
3901 gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg
3961 cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg
4021 ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac
4081 aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg
4141 aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct
4201 acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg
4261 cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg
4321 taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag
4381 cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt
4441 tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac
4501 cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat
4561 aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag
4621 gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg
4681 gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt
4741 gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg
4801 gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc
4861 ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca
4921 gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt
4981 ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc
5041 cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg
5101 cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta
5161 ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac
5221 aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc
5281 gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac
5341 gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt
5401 gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc
5461 gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg
5521 ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc
5581 ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa
5641 cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg
5701 tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta
5761 gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag
5821 cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg
5881 gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg
5941 aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa
6001 aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg
6061 gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt
6121 cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc
6181 tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg
6241 ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg
6301 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
6361 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
6421 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
6481 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
6541 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
6601 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
6661 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
6721 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
6781 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
6841 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
6901 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
6961 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
7021 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
7081 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
7141 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
7201 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
7261 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
7321 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
7381 tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata
7441 taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca
7501 tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac
7561 ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca
7621 aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc
7681 gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt
7741 tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg
7801 tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac
7861 tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa
7921 aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc
7981 aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc
8041 acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt
8101 tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg
8161 tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc
8221 cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact
8281 ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt
8341 tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat
8401 cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc
8461 gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa
8521 agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt
8581 atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag
8641 gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt
8701 tttaatgtac tgaattaacg ccgaattaat tcgggggatc tggattttag tactggattt
8761 tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa atacatacta
8821 agggtttctt atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatc
8881 tgggaactac tcacacatta ttatggagaa actcgagtca aatctcggtg acgggcagga
8941 ccggacgggg cggtaccggc aggctgaagt ccagctgcca gaaacccacg tcatgccagt
9001 tcccgtgctt gaagccggcc gcccgcagca tgccgcgggg ggcatatccg agcgcctcgt
9061 gcatgcgcac gctcgggtcg ttgggcagcc cgatgacagc gaccacgctc ttgaagccct
9121 gtgcctccag ggacttcagc aggtgggtgt agagcgtgga gcccagtccc gtccgctggt
9181 ggcgggggga gacgtacacg gtcgactcgg ccgtccagtc gtaggcgttg cgtgccttcc
9241 aggggcccgc gtaggcgatg ccggcgacct cgccgtccac ctcggcgacg agccagggat
9301 agcgctcccg cagacggacg aggtcgtccg tccactcctg cggttcctgc ggctcggtac
9361 ggaagttgac cgtgcttgtc tcgatgtagt ggttgacgat ggtgcagacc gccggcatgt
9421 ccgcctcggt ggcacggcgg atgtcggccg ggcgtcgttc tgggctcatg gtagactcga
9481 gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca aatgaaatga
9541 acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg
9601 tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt
9661 tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct
9721 tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc ttccttttct
9781 actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag gtttcccgat
9841 attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgata
9901 ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc cacttgcttt
9961 gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct
10021 ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc gcaatgatgg
10081 catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca gatagctggg
10141 caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc aatagccctt
10201 tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc gtgctccacc
10261 atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
10321 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
10381 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
10441 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
10501 attacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc
10561 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
10621 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
10681 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag cttgagcttg
10741 gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat tcaaatagag
10801 gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct cttacgactc
10861 aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt ctactccaaa
10921 aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta
10981 atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata
11041 gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt
11101 gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg
11161 gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact
11221 gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga
11281 agttcatttc atttggagag aacacggggg actcttgac
//
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
| P2376/pECMV-3×FLAG-MRP4(2364-3837bp)人源基因质粒 |
| P2377/pEGFP-C3-ATG5人源基因质粒 |
| P2412/pECMV-3×FLAG-MAPK15人源基因质粒 |
| P2413/pECMV-3×FLAG-MS4A3人源基因质粒 |
| P2414/pECMV-3×FLAG-C19orf36人源基因质粒 |
| P2415/pECMV-3×FLAG-TIGIT人源基因质粒 |
| P2416/pECMV-3×FLAG-STXBP1人源基因质粒 |
| P2417/pECMV-3×FLAG-HDAC7人源基因质粒 |
| P2418/pECMV-3×FLAG-CXCR4人源基因质粒 |
| P2961/pECMV-3×FLAG-GUCY2C人源基因质粒 |
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pCAMBIA3301植物表达质粒
￥100 - 1000
