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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：
pGBKT7-GW
- 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：pGBKT7-GW酵母双杂交质粒
别称: pGBKT7-GW
质粒属性
质粒简介
pGBKT7-GW质粒是一个用GateWay方法做克隆的酵母双杂交质粒。
质粒图谱
质粒序列
LOCUS Exported 8981 bp ds-DNA circular SYN 11-OCT-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8981)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8981
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 30..734
/gene="S. cerevisiae ADH1"
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 762..1202
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/label=GAL4 DNA binding domain
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter 1213..1231
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1251..1280
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
protein_bind 1287..1411
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 1436..1466
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 1520..2179
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 2521..2826
/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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(2867..2991)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
terminator 3015..3062
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
terminator 3089..3276
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
primer_bind complement(3300..3316)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3324..3340
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
promoter complement(3348..3378)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3393..3414
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3702..4290)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
polyA_signal 4619..4666
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
CDS complement(4899..5693)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Kan
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5694..5798)
/gene="bla"
/label=AmpR promoter
rep_origin 5825..7167
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind complement(6788..6835)
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 7426..7707
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
CDS 7708..8382
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(8485..8940)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
ORIGIN
1 cggtgcgggc ctcttcgcta ttacgccaga tccttttgtt gtttccgggt gtacaatatg
61 gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata ccttcgttgg
121 tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac cagacaagac
181 ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg tacataacga
241 actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca ctaccctttt
301 tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct ttttttttct
361 tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaaa tgatggaaga
421 cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg
481 atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct
541 ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaagtt
601 tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt ttcctcgtca
661 ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa gctataccaa
721 gcatacaatc aactccaagc ttgaagcaag cctcctgaaa gatgaagcta ctgtcttcta
781 tcgaacaagc atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga
841 agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt
901 ctccgctgac tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc
961 tatttctact gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac
1021 aggatataaa agcattgtta acaggattat ttgtacaaga taatgtgaat aaagatgccg
1081 tcacagatag attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa
1141 taagtgcgac atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat
1201 cgccggaatt tgtaatacga ctcactatag ggcgagccgc catcatggag gagcagaagc
1261 tgatctcaga ggaggacctg catatgacaa gtttgtacaa aaaagctgaa cgagaaacgt
1321 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
1381 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
1441 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
1501 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
1561 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
1621 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
1681 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
1741 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
1801 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
1861 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
1921 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
1981 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
2041 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
2101 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
2161 gtggcagggc ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc
2221 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
2281 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
2341 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
2401 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
2461 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
2521 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
2581 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
2641 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
2701 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
2761 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
2821 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat
2881 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
2941 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg tctcgacctg
3001 cagcggccgc ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
3061 tgcgcgcttg cagccaagct aattccgggc gaatttctta tgatttatga tttttattat
3121 taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa
3181 acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata
3241 gcatgaggtc gctcttattg accacacctc taccggcatg caagcttggc gtaatcatgg
3301 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
3361 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
3421 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
3481 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
3541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
3601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
3661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
3721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
3781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
3841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
3901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
3961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
4021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
4081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
4141 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
4201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
4261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
4321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
4381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
4441 gagtaacctg aggctatggc agggcctgcc gccccgacgt tggctgcgag ccctgggcct
4501 tcacccgaac ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta ttggccccaa
4561 tggggtctcg gtggggtatc gacagagtgc cagccctggg accgaacccc gcgtttatga
4621 acaaacgacc caacaccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt
4681 ccttccggta ttgtctcctt ccgtgtttca gttagcctcc ccctagggtg ggcgaagaac
4741 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
4801 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
4861 gtcgcttggt cggttcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
4921 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
4981 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
5041 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
5101 accatgatat tcggcaagca ggcatcgtca tgggtcacga cgagatcctc gccgtcgggc
5161 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
5221 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
5281 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
5341 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
5401 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
5461 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca
5521 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
5581 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
5641 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catactcttc
5701 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
5761 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
5821 cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat
5881 ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct
5941 attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg
6001 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga
6061 gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga
6121 gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat
6181 aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact
6241 ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt
6301 actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac
6361 cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg
6421 tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt
6481 tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc
6541 taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc
6601 aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa
6661 gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac
6721 agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa
6781 gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc
6841 gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt
6901 tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat
6961 agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta
7021 gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc
7081 actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa
7141 tgctatcatt tcctttgata ttggatcata ttaagaaacc attattatca tgacattaac
7201 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
7261 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
7321 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
7381 ctatgcggca tcagagcaga ttgtactgag agtgcaccat agatcaacga cattactata
7441 tatataatat aggaagcatt taatagaaca gcatcgtaat atatgtgtac tttgcagtta
7501 tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg tcaccttacg
7561 tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc ggtcgaaaaa
7621 agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg agtatacgtg
7681 attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg tagttctggt
7741 ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc tctagattcc
7801 gatgctgact tgctgggtat tatatgtgtg cccaatagaa agagaacaat tgacccggtt
7861 attgcaagga aaatttcaag tcttgtaaaa gcatataaaa atagttcagg cactccgaaa
7921 tacttggttg gcgtgtttcg taatcaacct aaggaggatg ttttggctct ggtcaatgat
7981 tacggcattg atatcgtcca actgcatgga gatgagtcgt ggcaagaata ccaagagttc
8041 ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat actactcagt
8101 gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc aggtgggaca
8161 ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga gagccccgaa
8221 agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga tgcgcttaga
8281 ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg tgtaaaagac
8341 tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac tgagtagtat
8401 ttatttaagt attgtttgtg cacttgccga tctatgcggt gtgaaatacc gcacagatgc
8461 gtaaggagaa aataccgcat caggaaattg taaacgttaa tattttgtta aaattcgcgt
8521 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
8581 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
8641 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
8701 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
8761 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
8821 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
8881 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
8941 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga t
//
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：pGBKT7-GW酵母双杂交质粒
别称: pGBKT7-GW
| 启动子: | ADH1 |
|---|---|
| 复制子: | 2U |
| 质粒大小: | 8981bp |
| 原核抗性: | Kan |
| 筛选标记: | TRP1 |
| 克隆菌株: | DB3.1 |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 杂交 |
| 基因种属： | 空载体 |
| 基因类型： | ORF,GateWay |
| 原核抗性： | Kan |
| 筛选标记： | TRP1 |
| 荧光蛋白： |
质粒简介
pGBKT7-GW质粒是一个用GateWay方法做克隆的酵母双杂交质粒。
质粒图谱
质粒序列
LOCUS Exported 8981 bp ds-DNA circular SYN 11-OCT-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8981)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8981
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 30..734
/gene="S. cerevisiae ADH1"
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 762..1202
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/label=GAL4 DNA binding domain
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter 1213..1231
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1251..1280
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
protein_bind 1287..1411
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 1436..1466
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 1520..2179
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 2521..2826
/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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(2867..2991)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
terminator 3015..3062
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
terminator 3089..3276
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
primer_bind complement(3300..3316)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3324..3340
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
promoter complement(3348..3378)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3393..3414
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3702..4290)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
polyA_signal 4619..4666
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
CDS complement(4899..5693)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Kan
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5694..5798)
/gene="bla"
/label=AmpR promoter
rep_origin 5825..7167
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind complement(6788..6835)
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 7426..7707
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
CDS 7708..8382
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(8485..8940)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
ORIGIN
1 cggtgcgggc ctcttcgcta ttacgccaga tccttttgtt gtttccgggt gtacaatatg
61 gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata ccttcgttgg
121 tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac cagacaagac
181 ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg tacataacga
241 actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca ctaccctttt
301 tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct ttttttttct
361 tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaaa tgatggaaga
421 cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg
481 atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct
541 ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaagtt
601 tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt ttcctcgtca
661 ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa gctataccaa
721 gcatacaatc aactccaagc ttgaagcaag cctcctgaaa gatgaagcta ctgtcttcta
781 tcgaacaagc atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga
841 agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt
901 ctccgctgac tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc
961 tatttctact gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac
1021 aggatataaa agcattgtta acaggattat ttgtacaaga taatgtgaat aaagatgccg
1081 tcacagatag attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa
1141 taagtgcgac atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat
1201 cgccggaatt tgtaatacga ctcactatag ggcgagccgc catcatggag gagcagaagc
1261 tgatctcaga ggaggacctg catatgacaa gtttgtacaa aaaagctgaa cgagaaacgt
1321 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
1381 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
1441 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
1501 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
1561 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
1621 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
1681 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
1741 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
1801 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
1861 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
1921 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
1981 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
2041 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
2101 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
2161 gtggcagggc ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc
2221 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
2281 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
2341 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
2401 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
2461 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
2521 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
2581 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
2641 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
2701 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
2761 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
2821 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat
2881 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
2941 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg tctcgacctg
3001 cagcggccgc ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
3061 tgcgcgcttg cagccaagct aattccgggc gaatttctta tgatttatga tttttattat
3121 taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa
3181 acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata
3241 gcatgaggtc gctcttattg accacacctc taccggcatg caagcttggc gtaatcatgg
3301 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
3361 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
3421 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
3481 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
3541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
3601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
3661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
3721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
3781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
3841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
3901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
3961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
4021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
4081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
4141 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
4201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
4261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
4321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
4381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
4441 gagtaacctg aggctatggc agggcctgcc gccccgacgt tggctgcgag ccctgggcct
4501 tcacccgaac ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta ttggccccaa
4561 tggggtctcg gtggggtatc gacagagtgc cagccctggg accgaacccc gcgtttatga
4621 acaaacgacc caacaccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt
4681 ccttccggta ttgtctcctt ccgtgtttca gttagcctcc ccctagggtg ggcgaagaac
4741 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
4801 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
4861 gtcgcttggt cggttcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
4921 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
4981 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
5041 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
5101 accatgatat tcggcaagca ggcatcgtca tgggtcacga cgagatcctc gccgtcgggc
5161 atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
5221 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
5281 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
5341 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
5401 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
5461 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca
5521 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
5581 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
5641 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catactcttc
5701 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
5761 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
5821 cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat
5881 ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct
5941 attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg
6001 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga
6061 gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga
6121 gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat
6181 aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact
6241 ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt
6301 actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac
6361 cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg
6421 tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt
6481 tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc
6541 taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc
6601 aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa
6661 gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac
6721 agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa
6781 gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc
6841 gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt
6901 tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat
6961 agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta
7021 gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc
7081 actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa
7141 tgctatcatt tcctttgata ttggatcata ttaagaaacc attattatca tgacattaac
7201 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
7261 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
7321 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
7381 ctatgcggca tcagagcaga ttgtactgag agtgcaccat agatcaacga cattactata
7441 tatataatat aggaagcatt taatagaaca gcatcgtaat atatgtgtac tttgcagtta
7501 tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg tcaccttacg
7561 tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc ggtcgaaaaa
7621 agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg agtatacgtg
7681 attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg tagttctggt
7741 ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc tctagattcc
7801 gatgctgact tgctgggtat tatatgtgtg cccaatagaa agagaacaat tgacccggtt
7861 attgcaagga aaatttcaag tcttgtaaaa gcatataaaa atagttcagg cactccgaaa
7921 tacttggttg gcgtgtttcg taatcaacct aaggaggatg ttttggctct ggtcaatgat
7981 tacggcattg atatcgtcca actgcatgga gatgagtcgt ggcaagaata ccaagagttc
8041 ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat actactcagt
8101 gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc aggtgggaca
8161 ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga gagccccgaa
8221 agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga tgcgcttaga
8281 ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg tgtaaaagac
8341 tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac tgagtagtat
8401 ttatttaagt attgtttgtg cacttgccga tctatgcggt gtgaaatacc gcacagatgc
8461 gtaaggagaa aataccgcat caggaaattg taaacgttaa tattttgtta aaattcgcgt
8521 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
8581 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
8641 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
8701 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
8761 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
8821 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
8881 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
8941 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga t
//
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
| P2677/pENTR223-MBOAT2-C674A人源基因质粒 |
| P2678/pENTR223-MTUS2-A26G-C1039G人源基因质粒 |
| P2679/pENTR223-EIF3H-G6A-A1047人源基因质粒 |
| P2680/pENTR223-SETMAR（N端有缺失）人源基因质粒 |
| P2681/pENTR223-GMPPB人源基因质粒 |
| P2682/pENTR223-TRUB1人源基因质粒 |
| P2683/pENTR223-CHST11-C1045T-A1055C人源基因质粒 |
| P2684/pENTR223-UROD-G908T人源基因质粒 |
| P2685/pENTR223-RAD51AP1人源基因质粒 |
| P2686/pENTR223-ZFP28-G29A-C971T人源基因质粒 |
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文献和实验相关实验
相关专题 酵母双杂交技术专题 1.构建CaM诱饵质粒 ① PCR 扩增CaM基因的开放阅读框，正向和反向引物分别加接酶切位点。 ② 扩增得到的CaM插入T载体 ( T–CaM) 。 ③ T–CaM和pGBKT7 质粒 分别双酶切。 ④ 胶回收目的DNA。 ⑤ 将CaM连入pGBKT7，构建成表达载体 pGBKT-CaM，转化大肠杆菌DH5&alpha
表达的调控，单单靠GLA4不知道能不能起到调控蛋白表达的目的？有这方面的文献吗？先谢谢各位大侠的帮助了！！！！ shilei5794749 类似GAL4-UAS系统，楼主可以借鉴这个系统。GLA4可以用pGBKT7或者pBIND上的，做成删除NLS序列的GAL4(BD+AD)或者GAL4BD-VP16。此质粒还可融合你要研究的蛋白，上游可以加调控元件。 另一个质粒用UAS序列打头，后置报告基因就行啦！ 版主dnazyme留言:
开始做酵母双杂交了，计划用一个膜蛋白去筛库，已经做好了工作量大，而预期结果要靠点小运气的心理准备，面前真是一条很长的路要走！ 在丁香园学习了很久，希望能将我这次试验过程中遇到的困难和思索记录下来，跟大家一同讨论，共同学习。 今天先说说实验之前的试剂准备，为了保证实验能顺利进行，我选择了clontech的matchmaker gold系统，订购了人脑cDNA文库（标准化的），还有培养基也是订的商品化的，再有一个提酵母质粒的KIT 1，GOLD系统式对系统3的升级版，替换了一个报告
pGBKT7-GW酵母双杂交质粒
￥100 - 1000
