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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：
pBI121
- 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：pBI121①植物表达质粒
别称: pBI121①
质粒属性
质粒简介
pBI121载体是一个双元植物农杆菌表达载体，能够高效转染植物。该载体来源于pB221和Bin19载体。载体上含有从ColE1来源的ori复制元件，从CaMV中来源的pROK1元件，大肠杆菌新霉素磷酸转移酶II基因（Neomycin phosphotransferase II Gene, NptII），新霉素抗性基因(Neomycin resistant gene, Neo), 敏感基因Beta葡糖苷酸酶(glucuronidase)，Ter 农杆菌Ti 质粒胭脂氨酸合成酶原件等。pBI121载体是卡那抗性质粒。
质粒图谱
质粒序列
LOCUS Exported 14758 bp ds-DNA circular SYN 13-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14758)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..14758
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/note="traJ"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/note="TetR"
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
promoter 2602..2781
/note="NOS promoter"
/note="nopaline synthase promoter"
promoter 2634..2817
/function="NOS promoter"
/note="NOS promoter"
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 4823..4844
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4859..4889
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4897..4913
/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 4921..4937
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 5461..5806
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 5845..7656
/codon_start=1
/gene="uidA (or gusA) from E. coli"
/product="beta-glucuronidase"
/note="GUS"
/translation="MLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAI
AVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEV
MEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHD
FFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQ
VVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQF
LINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDW
ADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARD
KNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISD
LFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMY
TDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRK
PKSAAFLLQKRWTGMNFGEKPQQGGKQ"
terminator 7727..7979
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(7988..8004)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 8622..8646
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(10242..11390)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/note="trfA"
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(11689..12483)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 12675..13442
/mobile_element_type="insertion sequence:IS1"
/note="IS1"
/note="prokaryotic transposable element"
rep_origin 14065..18
/note="oriV"
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 ggatccccgg gtggtcagtc ccttatgtta cgtcctgtag aaaccccaac ccgtgaaatc
5881 aaaaaactcg acggcctgtg ggcattcagt ctggatcgcg aaaactgtgg aattgatcag
5941 cgttggtggg aaagcgcgtt acaagaaagc cgggcaattg ctgtgccagg cagttttaac
6001 gatcagttcg ccgatgcaga tattcgtaat tatgcgggca acgtctggta tcagcgcgaa
6061 gtctttatac cgaaaggttg ggcaggccag cgtatcgtgc tgcgtttcga tgcggtcact
6121 cattacggca aagtgtgggt caataatcag gaagtgatgg agcatcaggg cggctatacg
6181 ccatttgaag ccgatgtcac gccgtatgtt attgccggga aaagtgtacg tatcaccgtt
6241 tgtgtgaaca acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa
6301 aacggcaaga aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc
6361 agcgtaatgc tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat
6421 gtcgcgcaag actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc
6481 agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg
6541 actttgcaag tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg
6601 tgcgtcacag ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg
6661 tcagtggcag tgaagggcga acagttcctg attaaccaca aaccgttcta ctttactggc
6721 tttggtcgtc atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg
6781 cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct
6841 tacgctgaag agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact
6901 gctgctgtcg gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa
6961 gaactgtaca gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt
7021 aaagagctga tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac
7081 gaaccggata cccgtccgca aggtgcacgg gaatatttcg cgccactggc ggaagcaacg
7141 cgtaaactcg acccgacgcg tccgatcacc tgcgtcaatg taatgttctg cgacgctcac
7201 accgatacca tcagcgatct ctttgatgtg ctgtgcctga accgttatta cggatggtat
7261 gtccaaagcg gcgatttgga aacggcagag aaggtactgg aaaaagaact tctggcctgg
7321 caggagaaac tgcatcagcc gattatcatc accgaatacg gcgtggatac gttagccggg
7381 ctgcactcaa tgtacaccga catgtggagt gaagagtatc agtgtgcatg gctggatatg
7441 tatcaccgcg tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc
7501 gattttgcga cctcgcaagg catattgcgc gttggcggta acaagaaagg gatcttcact
7561 cgcgaccgca aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac tggcatgaac
7621 ttcggtgaaa aaccgcagca gggaggcaaa caatgaatca acaactctcc tggcgcacca
7681 tcgtcggcta cagcctcggg aattgctacc gagctcgaat ttccccgatc gttcaaacat
7741 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata
7801 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
7861 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa
7921 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg
7981 ggaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
8041 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
8101 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc
8161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
8221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
8281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
8341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
8401 tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa caggattttc
8461 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
8521 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa
8581 aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc
8641 ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat
8701 acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact
8761 ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac
8821 ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg
8881 atcaaatatc atctccctcg cagagatccg aattatcagc cttcttattc atttctcgct
8941 taaccgtgac aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggata
9001 aagccgctga ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatatcaac
9061 tcccctatcc attgctcacc gaatggtaca ggtcggggac ccgaagttcc gactgtcggc
9121 ctgatgcatc cccggctgat cgaccccaga tctggggctg agaaagccca gtaaggaaac
9181 aactgtaggt tcgagtcgcg agatcccccg gaaccaaagg aagtaggtta aacccgctcc
9241 gatcaggccg agccacgcca ggccgagaac attggttcct gtaggcatcg ggattggcgg
9301 atcaaacact aaagctactg gaacgagcag aagtcctccg gccgccagtt gccaggcggt
9361 aaaggtgagc agaggcacgg gaggttgcca cttgcgggtc agcacggttc cgaacgccat
9421 ggaaaccgcc cccgccaggc ccgctgcgac gccgacagga tctagcgctg cgtttggtgt
9481 caacaccaac agcgccacgc ccgcagttcc gcaaatagcc cccaggaccg ccatcaatcg
9541 tatcgggcta cctagcagag cggcagagat gaacacgacc atcagcggct gcacagcgcc
9601 taccgtcgcc gcgaccccgc ccggcaggcg gtagaccgaa ataaacaaca agctccagaa
9661 tagcgaaata ttaagtgcgc cgaggatgaa gatgcgcatc caccagattc ccgttggaat
9721 ctgtcggacg atcatcacga gcaataaacc cgccggcaac gcccgcagca gcataccggc
9781 gacccctcgg cctcgctgtt cgggctccac gaaaacgccg gacagatgcg ccttgtgagc
9841 gtccttgggg ccgtcctcct gtttgaagac cgacagccca atgatctcgc cgtcgatgta
9901 ggcgccgaat gccacggcat ctcgcaaccg ttcagcgaac gcctccatgg gctttttctc
9961 ctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggat
10021 ctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaat
10081 tgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcga
10141 tactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcg
10201 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc
10261 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct
10321 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg
10381 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt
10441 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca
10501 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca
10561 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg
10621 gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
10681 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc
10741 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca
10801 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa
10861 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca
10921 gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
10981 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg
11041 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata
11101 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacga
11161 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg
11221 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg
11281 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc
11341 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct
11401 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct
11461 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc
11521 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc
11581 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag
11641 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattc
11701 atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa
11761 aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc
11821 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt
11881 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc
11941 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc
12001 ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc
12061 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa
12121 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata
12181 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg
12241 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc
12301 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata
12361 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc
12421 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc
12481 catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat
12541 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca
12601 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg
12661 aaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga
12721 ggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtg
12781 gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa
12841 acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt
12901 gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc
12961 aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc
13021 cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta
13081 aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc
13141 acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct
13201 ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa
13261 agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga
13321 ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc
13381 atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta
13441 cccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtc
13501 catgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgc
13561 cttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatctta
13621 tcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtct
13681 tggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttt
13741 tcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccc
13801 cgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccg
13861 gccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccaccca
13921 ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggca
13981 cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcc
14041 cggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgagg
14101 ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg
14161 ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca
14221 aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac
14281 gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat
14341 aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat
14401 ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc
14461 cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc
14521 cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt
14581 gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc
14641 gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttct
14701 ggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccgg
//
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：pBI121①植物表达质粒
别称: pBI121①
| 启动子: | 35s |
|---|---|
| 复制子: | oriV |
| 终止子: | NOS |
| 质粒分类: | 植物系列，蛋白过表达载体 |
| 质粒大小: | 14758bp |
| 原核抗性: | Kan |
| 筛选标记: | G418 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 5'测序引物: | 35S:GACGCACAATCCCACTATCC |
| 3'测序引物: | 根据序列设计引物 |
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： | Neo/G418 |
| 荧光蛋白： | Gus |
质粒简介
pBI121载体是一个双元植物农杆菌表达载体，能够高效转染植物。该载体来源于pB221和Bin19载体。载体上含有从ColE1来源的ori复制元件，从CaMV中来源的pROK1元件，大肠杆菌新霉素磷酸转移酶II基因（Neomycin phosphotransferase II Gene, NptII），新霉素抗性基因(Neomycin resistant gene, Neo), 敏感基因Beta葡糖苷酸酶(glucuronidase)，Ter 农杆菌Ti 质粒胭脂氨酸合成酶原件等。pBI121载体是卡那抗性质粒。
质粒图谱
质粒序列
LOCUS Exported 14758 bp ds-DNA circular SYN 13-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14758)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..14758
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/note="traJ"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/note="TetR"
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
promoter 2602..2781
/note="NOS promoter"
/note="nopaline synthase promoter"
promoter 2634..2817
/function="NOS promoter"
/note="NOS promoter"
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 4823..4844
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4859..4889
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4897..4913
/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 4921..4937
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 5461..5806
/note="CaMV 35S promoter"
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 5845..7656
/codon_start=1
/gene="uidA (or gusA) from E. coli"
/product="beta-glucuronidase"
/note="GUS"
/translation="MLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAI
AVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEV
MEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHD
FFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQ
VVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQF
LINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDW
ADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARD
KNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISD
LFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMY
TDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRK
PKSAAFLLQKRWTGMNFGEKPQQGGKQ"
terminator 7727..7979
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(7988..8004)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 8622..8646
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(10242..11390)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/note="trfA"
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(11689..12483)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 12675..13442
/mobile_element_type="insertion sequence:IS1"
/note="IS1"
/note="prokaryotic transposable element"
rep_origin 14065..18
/note="oriV"
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 ggatccccgg gtggtcagtc ccttatgtta cgtcctgtag aaaccccaac ccgtgaaatc
5881 aaaaaactcg acggcctgtg ggcattcagt ctggatcgcg aaaactgtgg aattgatcag
5941 cgttggtggg aaagcgcgtt acaagaaagc cgggcaattg ctgtgccagg cagttttaac
6001 gatcagttcg ccgatgcaga tattcgtaat tatgcgggca acgtctggta tcagcgcgaa
6061 gtctttatac cgaaaggttg ggcaggccag cgtatcgtgc tgcgtttcga tgcggtcact
6121 cattacggca aagtgtgggt caataatcag gaagtgatgg agcatcaggg cggctatacg
6181 ccatttgaag ccgatgtcac gccgtatgtt attgccggga aaagtgtacg tatcaccgtt
6241 tgtgtgaaca acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa
6301 aacggcaaga aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc
6361 agcgtaatgc tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat
6421 gtcgcgcaag actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc
6481 agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg
6541 actttgcaag tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg
6601 tgcgtcacag ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg
6661 tcagtggcag tgaagggcga acagttcctg attaaccaca aaccgttcta ctttactggc
6721 tttggtcgtc atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg
6781 cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct
6841 tacgctgaag agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact
6901 gctgctgtcg gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa
6961 gaactgtaca gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt
7021 aaagagctga tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac
7081 gaaccggata cccgtccgca aggtgcacgg gaatatttcg cgccactggc ggaagcaacg
7141 cgtaaactcg acccgacgcg tccgatcacc tgcgtcaatg taatgttctg cgacgctcac
7201 accgatacca tcagcgatct ctttgatgtg ctgtgcctga accgttatta cggatggtat
7261 gtccaaagcg gcgatttgga aacggcagag aaggtactgg aaaaagaact tctggcctgg
7321 caggagaaac tgcatcagcc gattatcatc accgaatacg gcgtggatac gttagccggg
7381 ctgcactcaa tgtacaccga catgtggagt gaagagtatc agtgtgcatg gctggatatg
7441 tatcaccgcg tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc
7501 gattttgcga cctcgcaagg catattgcgc gttggcggta acaagaaagg gatcttcact
7561 cgcgaccgca aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac tggcatgaac
7621 ttcggtgaaa aaccgcagca gggaggcaaa caatgaatca acaactctcc tggcgcacca
7681 tcgtcggcta cagcctcggg aattgctacc gagctcgaat ttccccgatc gttcaaacat
7741 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata
7801 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
7861 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa
7921 aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg
7981 ggaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
8041 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
8101 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc
8161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
8221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
8281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
8341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
8401 tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa caggattttc
8461 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
8521 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa
8581 aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc
8641 ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat
8701 acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact
8761 ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac
8821 ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg
8881 atcaaatatc atctccctcg cagagatccg aattatcagc cttcttattc atttctcgct
8941 taaccgtgac aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggata
9001 aagccgctga ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatatcaac
9061 tcccctatcc attgctcacc gaatggtaca ggtcggggac ccgaagttcc gactgtcggc
9121 ctgatgcatc cccggctgat cgaccccaga tctggggctg agaaagccca gtaaggaaac
9181 aactgtaggt tcgagtcgcg agatcccccg gaaccaaagg aagtaggtta aacccgctcc
9241 gatcaggccg agccacgcca ggccgagaac attggttcct gtaggcatcg ggattggcgg
9301 atcaaacact aaagctactg gaacgagcag aagtcctccg gccgccagtt gccaggcggt
9361 aaaggtgagc agaggcacgg gaggttgcca cttgcgggtc agcacggttc cgaacgccat
9421 ggaaaccgcc cccgccaggc ccgctgcgac gccgacagga tctagcgctg cgtttggtgt
9481 caacaccaac agcgccacgc ccgcagttcc gcaaatagcc cccaggaccg ccatcaatcg
9541 tatcgggcta cctagcagag cggcagagat gaacacgacc atcagcggct gcacagcgcc
9601 taccgtcgcc gcgaccccgc ccggcaggcg gtagaccgaa ataaacaaca agctccagaa
9661 tagcgaaata ttaagtgcgc cgaggatgaa gatgcgcatc caccagattc ccgttggaat
9721 ctgtcggacg atcatcacga gcaataaacc cgccggcaac gcccgcagca gcataccggc
9781 gacccctcgg cctcgctgtt cgggctccac gaaaacgccg gacagatgcg ccttgtgagc
9841 gtccttgggg ccgtcctcct gtttgaagac cgacagccca atgatctcgc cgtcgatgta
9901 ggcgccgaat gccacggcat ctcgcaaccg ttcagcgaac gcctccatgg gctttttctc
9961 ctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggat
10021 ctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaat
10081 tgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcga
10141 tactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcg
10201 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc
10261 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct
10321 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg
10381 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt
10441 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca
10501 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca
10561 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg
10621 gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
10681 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc
10741 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca
10801 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa
10861 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca
10921 gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
10981 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg
11041 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata
11101 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacga
11161 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg
11221 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg
11281 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc
11341 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct
11401 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct
11461 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc
11521 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc
11581 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag
11641 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattc
11701 atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa
11761 aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc
11821 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt
11881 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc
11941 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc
12001 ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc
12061 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa
12121 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata
12181 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg
12241 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc
12301 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata
12361 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc
12421 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc
12481 catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat
12541 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca
12601 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg
12661 aaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga
12721 ggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtg
12781 gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa
12841 acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt
12901 gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc
12961 aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc
13021 cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta
13081 aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc
13141 acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct
13201 ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa
13261 agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga
13321 ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc
13381 atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta
13441 cccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtc
13501 catgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgc
13561 cttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatctta
13621 tcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtct
13681 tggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttt
13741 tcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccc
13801 cgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccg
13861 gccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccaccca
13921 ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggca
13981 cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcc
14041 cggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgagg
14101 ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg
14161 ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca
14221 aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac
14281 gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat
14341 aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat
14401 ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc
14461 cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc
14521 cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt
14581 gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc
14641 gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttct
14701 ggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccgg
//
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
| P2395/pENTR223-SIAT7E人源基因质粒 |
| P2396/pENTR223-AATF人源基因质粒 |
| P2397/pENTR223-SMN2-aa225缺失人源基因质粒 |
| P2398/pENTR223-HAX1-C16G人源基因质粒 |
| P2399/pENTR223-BUD23-A833人源基因质粒 |
| P2400/pENTR223-EMC2-A24G-13C人源基因质粒 |
| P2401/pENTR223-PEF1人源基因质粒 |
| P2402/pENTR223-INKA2人源基因质粒 |
| P2403/PENTR223-TSGA13（可引物突变）人源基因质粒 |
| P2404/pENTR223-C5AR1人源基因质粒 |
| P2405/pENTR223-DFFA-C871T人源基因质粒 |
| P2406/pENTR223-LRFN4(934-1908bp)人源基因质粒 |
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文献和实验相关实验
。 GhVP 基因的亚细胞定位 将构建好的 pBI121-GhVP∷GFP 载体用金粉包裹，把金粉包裹好的质粒加入基因枪 GDS-80 轰击洋葱内表皮细胞， 在激光共聚焦显微镜下观察。可以看出，空载体 pBI121-GFP 转化洋葱表皮后， 在整个洋葱细胞均能看到绿色荧光。pBI121-GhVP∷GFP 转化洋葱后，VP 蛋白定位于洋葱表皮细胞的细胞膜上，在玻片上加蔗糖，待质壁分离后，GhVP 蛋白在洋葱表皮细胞的细胞膜和细胞间质中均有分布（图3）。结果表明 GhVP 蛋白是 1 个跨膜蛋白， 与细胞
121 在文献"香蕉 Maasr1 基因 RNAi 植物表达载体的构建及鉴定.热带作物学 报， 2009 年， 30 (3) :333-337. "中公开过，公众可从中国农业科学院棉花研究所抗逆鉴定课 题组实验室获得。基因枪为美国 WEALTEC 的基因枪 GDS-80 。非抗虫棉花材料沪 749513 在文献"高频体细胞胚胎发生的优异棉花种质材料筛选.分子植物育种， 2012 ，10 (6) ，683-688. "中公开过，公众可从中国农业科学院棉花研究 所抗逆鉴定课题组实验室获得。 实施例
质粒是存在于细菌染色体外的一个或多个能独立复制并稳定遗传的小型环状双链DNA分子，其分子量一般在0. 2～10kD 范围内。由于质粒分子小，便于分离和提取，可以携带目的基因进入细菌、动物细胞或植物体内进行扩增与表达。基因克隆实验中常将经改造后的质粒作为运送基因的载体，质粒D2NA提取效率及质量对后续实验步骤(如酶切、连接、转化大肠杆菌与PCR扩增等)的成功与否有着直接的关系，因而高效快速地从细菌细胞中分离纯化质粒具有重要意义[ 1 ] 。目前从细菌中提取质粒DNA多采用碱裂解法，该法操作简便
pBI121①植物表达质粒
￥100 - 1000
