| 出品公司: | |
|---|---|
| 载体名称: | pET-12c |
| 质粒类型: | 大肠杆菌表达载体 |
| 高拷贝/低拷贝: | 低拷贝 |
| 克隆方法: | 多克隆位点；限制性内切酶 |
| 启动子: | T7/ lacI |
| 载体大小: | 4675 bp |
| 5' 测序引物及序列: | T7 Fwd: 5'd[TAATACGACTCACTATAGGG]3' |
| 3' 测序引物及序列: | -- |
| 载体标签: | ompT Tag (N-端) |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 克隆菌株: | DH5a |
| 表达菌株: | BL21系列 |
| 备注: | pET-12a，b，c仅阅读框（读码框）不同，彼此相差1个bp。 |
| 产品目录号: | ZY71553-3 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 诱导型（IPTG) |
| 病毒/非病毒: | 非病毒 |
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pET-12c(+)
- 库存：
20
- 供应商：
泽叶生物
pet12c载体基本信息
pet12c载体质粒图谱和多克隆位点信息
| Feature Name | Start | End | |
|---|---|---|---|
| tet (w/ gaps) | 86 | 1588 | |
| T7_terminator | 507 | 379 | |
| T7_Terminal_primer | 447 | 465 | |
| T7_transl_en_RBS | 607 | 591 | |
| T7_promoter | 662 | 644 | |
| pBRrevBam_primer | 765 | 746 | |
| ROP | 2227 | 2418 | |
| pGEX_3_primer | 2434 | 2412 | |
| Ampicillin | 4467 | 3607 | |
| AmpR_promoter | 4537 | 4509 |
| ORF | Start | End | |
|---|---|---|---|
| ORF frame 1 | 799 | 1386 | |
| ORF frame 1 | 1393 | 908 | |
| ORF frame 2 | 971 | 1588 | |
| ORF frame 2 | 4467 | 3607 |
| Enzyme Name | Cut |
|---|---|
| ClaI | 24 |
| HindIII | 29 |
| NheI | 229 |
| BamHI | 510 |
| SalI | 518 |
| NdeI | 581 |
| XbaI | 619 |
| BglII | 677 |
| EagI | 1251 |
| MscI | 1758 |
| PvuII | 2378 |
| PstI | 3925 |
| AatII | 4602 |
| EcoRI | 4673 |
pet12c载体简介
pet12c载体序列
LOCUS pET12c 4675 bp DNA circular SYN
DEFINITION pET12c
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTOAUTHORAD1|Novagen Inc.|
FEATURES Location/Qualifiers
source 1..4675
/organism="pET12c"
/mol_type="other DNA"
gene 86..1588
/label="tet (w/ gaps)"
/gene="tet (w/ gaps)"
terminator complement(379..507)
/label="T7_terminator"
misc_feature 447..465
/label="T7_Terminal_primer"
misc_feature complement(591..607)
/label="T7_transl_en_RBS"
promoter complement(644..662)
/label="T7_promoter"
misc_feature complement(746..765)
/label="pBRrevBam_primer"
CDS 799..1386
/label="ORF frame 1"
/translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCF
LMQESHKGERRSTDALESLQPSQLLPVGAGHDYRRRTYDCLLYHATRRTGAGSALGHF
RRGPLSLERDDDRPVACGIRNLARPRSSLRHWSRHQTFRREAGHYRRHGGRRAGLRLA
GVRDARLDGLPHYDSSRFRRHRDARVAGHAVQAGR*"
CDS complement(908..1393)
/label="ORF frame 1"
/translation="MVVIYLPGQHGLQRGHPDAAGSEKNHNGEGHPASRRERQQDVAQ
RVGRHAGDNGLLLAETFGGGTSDEGLSEGVQDSEYRKRQADHRRAPAKAVLAENDPER
CRHLSYELHDKEDSHKCGDDSHAPRPPEGADWVEGSQGHRSIDALPYATPALGSSPVV
G*"
CDS 971..1588
/label="ORF frame 2"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
misc_feature 2227..2418
/label="ROP"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
misc_feature complement(2412..2434)
/label="pGEX_3_primer"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
gene complement(3607..4467)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
CDS complement(3607..4467)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(4509..4537)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT CACAGTTAAA
61 TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA TCATGGCGAC
361 CACACCCGTC CTGTGGATAT CCGGATATAG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA
421 CCCGTTTAGA GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT
481 CAGCTTCCTT TCGGGCTTTG TTAGCAGCCG GATCCCCGTC GACGCAAAAG AGCTGATCGC
541 GATAGGGGTT GTCAGGACTA TTCCTAGGAG TTTCGCCCGC ATATGTATAT CTCCTTCTTA
601 AAGTTAAACA AAATTATTTC TAGAGGGAAA CCGTTGTGGT CTCCCTATAG TGAGTCGTAT
661 TAATTTCGCG GGATCGAGAT CTCGATCCTC TACGCCGGAC GCATCGTGGC CGGCATCACC
721 GGCGCCACAG GTGCGGTTGC TGGCGCCTAT ATCGCCGACA TCACCGATGG GGAAGATCGG
781 GCTCGCCACT TCGGGCTCAT GAGCGCTTGT TTCGGCGTGG GTATGGTGGC AGGCCCCGTG
841 GCCGGGGGAC TGTTGGGCGC CATCTCCTTG CATGCACCAT TCCTTGCGGC GGCGGTGCTC
901 AACGGCCTCA ACCTACTACT GGGCTGCTTC CTAATGCAGG AGTCGCATAA GGGAGAGCGT
961 CGATCGACCG ATGCCCTTGA GAGCCTTCAA CCCAGTCAGC TCCTTCCGGT GGGCGCGGGG
1021 CATGACTATC GTCGCCGCAC TTATGACTGT CTTCTTTATC ATGCAACTCG TAGGACAGGT
1081 GCCGGCAGCG CTCTGGGTCA TTTTCGGCGA GGACCGCTTT CGCTGGAGCG CGACGATGAT
1141 CGGCCTGTCG CTTGCGGTAT TCGGAATCTT GCACGCCCTC GCTCAAGCCT TCGTCACTGG
1201 TCCCGCCACC AAACGTTTCG GCGAGAAGCA GGCCATTATC GCCGGCATGG CGGCCGACGC
1261 GCTGGGCTAC GTCTTGCTGG CGTTCGCGAC GCGAGGCTGG ATGGCCTTCC CCATTATGAT
1321 TCTTCTCGCT TCCGGCGGCA TCGGGATGCC CGCGTTGCAG GCCATGCTGT CCAGGCAGGT
1381 AGATGACGAC CATCAGGGAC AGCTTCAAGG ATCGCTCGCG GCTCTTACCA GCCTAACTTC
1441 GATCACTGGA CCGCTGATCG TCACGGCGAT TTATGCCGCC TCGGCGAGCA CATGGAACGG
1501 GTTGGCATGG ATTGTAGGCG CCGCCCTATA CCTTGTCTGC CTCCCCGCGT TGCGTCGCGG
1561 TGCATGGAGC CGGGCCACCT CGACCTGAAT GGAAGCCGGC GGCACCTCGC TAACGGATTC
1621 ACCACTCCAA GAATTGGAGC CAATCAATTC TTGCGGAGAA CTGTGAATGC GCAAACCAAC
1681 CCTTGGCAGA ACATATCCAT CGCGTCCGCC ATCTCCAGCA GCCGCACGCG GCGCATCTCG
1741 GGCAGCGTTG GGTCCTGGCC ACGGGTGCGC ATGATCGTGC TCCTGTCGTT GAGGACCCGG
1801 CTAGGCTGGC GGGGTTGCCT TACTGGTTAG CAGAATGAAT CACCGATACG CGAGCGAACG
1861 TGAAGCGACT GCTGCTGCAA AACGTCTGCG ACCTGAGCAA CAACATGAAT GGTCTTCGGT
1921 TTCCGTGTTT CGTAAAGTCT GGAAACGCGG AAGTCAGCGC CCTGCACCAT TATGTTCCGG
1981 ATCTGCATCG CAGGATGCTG CTGGCTACCC TGTGGAACAC CTACATCTGT ATTAACGAAG
2041 CGCTGGCATT GACCCTGAGT GATTTTTCTC TGGTCCCGCC GCATCCATAC CGCCAGTTGT
2101 TTACCCTCAC AACGTTCCAG TAACCGGGCA TGTTCATCAT CAGTAACCCG TATCGTGAGC
2161 ATCCTCTCTC GTTTCATCGG TATCATTACC CCCATGAACA GAAATCCCCC TTACACGGAG
2221 GCATCAGTGA CCAAACAGGA AAAAACCGCC CTTAACATGG CCCGCTTTAT CAGAAGCCAG
2281 ACATTAACGC TTCTGGAGAA ACTCAACGAG CTGGACGCGG ATGAACAGGC AGACATCTGT
2341 GAATCGCTTC ACGACCACGC TGATGAGCTT TACCGCAGCT GCCTCGCGCG TTTCGGTGAT
2401 GACGGTGAAA ACCTCTGACA CATGCAGCTC CCGGAGACGG TCACAGCTTG TCTGTAAGCG
2461 GATGCCGGGA GCAGACAAGC CCGTCAGGGC GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC
2521 GCAGCCATGA CCCAGTCACG TAGCGATAGC GGAGTGTATA CTGGCTTAAC TATGCGGCAT
2581 CAGAGCAGAT TGTACTGAGA GTGCACCATA TATGCGGTGT GAAATACCGC ACAGATGCGT
2641 AAGGAGAAAA TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC
2701 GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC
2761 AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA
2821 CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA
2881 CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC
2941 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA
3001 CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA
3061 TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA
3121 GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA
3181 CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG
3241 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA CAGTATTTGG
3301 TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG
3361 CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG
3421 AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA
3481 CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT
3541 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC
3601 TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC
3661 ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC
3721 TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC
3781 AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC
3841 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT
3901 GCGCAACGTT GTTGCCATTG CTGCAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC
3961 TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA
4021 AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT
4081 ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG
4141 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC
4201 GAGTTGCTCT TGCCCGGCGT CAACACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA
4261 AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT
4321 GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT
4381 CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG
4441 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA
4501 TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT
4561 AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT
4621 CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTTC AAGAA
DEFINITION pET12c
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTOAUTHORAD1|Novagen Inc.|
FEATURES Location/Qualifiers
source 1..4675
/organism="pET12c"
/mol_type="other DNA"
gene 86..1588
/label="tet (w/ gaps)"
/gene="tet (w/ gaps)"
terminator complement(379..507)
/label="T7_terminator"
misc_feature 447..465
/label="T7_Terminal_primer"
misc_feature complement(591..607)
/label="T7_transl_en_RBS"
promoter complement(644..662)
/label="T7_promoter"
misc_feature complement(746..765)
/label="pBRrevBam_primer"
CDS 799..1386
/label="ORF frame 1"
/translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCF
LMQESHKGERRSTDALESLQPSQLLPVGAGHDYRRRTYDCLLYHATRRTGAGSALGHF
RRGPLSLERDDDRPVACGIRNLARPRSSLRHWSRHQTFRREAGHYRRHGGRRAGLRLA
GVRDARLDGLPHYDSSRFRRHRDARVAGHAVQAGR*"
CDS complement(908..1393)
/label="ORF frame 1"
/translation="MVVIYLPGQHGLQRGHPDAAGSEKNHNGEGHPASRRERQQDVAQ
RVGRHAGDNGLLLAETFGGGTSDEGLSEGVQDSEYRKRQADHRRAPAKAVLAENDPER
CRHLSYELHDKEDSHKCGDDSHAPRPPEGADWVEGSQGHRSIDALPYATPALGSSPVV
G*"
CDS 971..1588
/label="ORF frame 2"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
misc_feature 2227..2418
/label="ROP"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
misc_feature complement(2412..2434)
/label="pGEX_3_primer"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
gene complement(3607..4467)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVI
FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL
LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG
PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
CDS complement(3607..4467)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(4509..4537)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT CACAGTTAAA
61 TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA TCATGGCGAC
361 CACACCCGTC CTGTGGATAT CCGGATATAG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA
421 CCCGTTTAGA GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT
481 CAGCTTCCTT TCGGGCTTTG TTAGCAGCCG GATCCCCGTC GACGCAAAAG AGCTGATCGC
541 GATAGGGGTT GTCAGGACTA TTCCTAGGAG TTTCGCCCGC ATATGTATAT CTCCTTCTTA
601 AAGTTAAACA AAATTATTTC TAGAGGGAAA CCGTTGTGGT CTCCCTATAG TGAGTCGTAT
661 TAATTTCGCG GGATCGAGAT CTCGATCCTC TACGCCGGAC GCATCGTGGC CGGCATCACC
721 GGCGCCACAG GTGCGGTTGC TGGCGCCTAT ATCGCCGACA TCACCGATGG GGAAGATCGG
781 GCTCGCCACT TCGGGCTCAT GAGCGCTTGT TTCGGCGTGG GTATGGTGGC AGGCCCCGTG
841 GCCGGGGGAC TGTTGGGCGC CATCTCCTTG CATGCACCAT TCCTTGCGGC GGCGGTGCTC
901 AACGGCCTCA ACCTACTACT GGGCTGCTTC CTAATGCAGG AGTCGCATAA GGGAGAGCGT
961 CGATCGACCG ATGCCCTTGA GAGCCTTCAA CCCAGTCAGC TCCTTCCGGT GGGCGCGGGG
1021 CATGACTATC GTCGCCGCAC TTATGACTGT CTTCTTTATC ATGCAACTCG TAGGACAGGT
1081 GCCGGCAGCG CTCTGGGTCA TTTTCGGCGA GGACCGCTTT CGCTGGAGCG CGACGATGAT
1141 CGGCCTGTCG CTTGCGGTAT TCGGAATCTT GCACGCCCTC GCTCAAGCCT TCGTCACTGG
1201 TCCCGCCACC AAACGTTTCG GCGAGAAGCA GGCCATTATC GCCGGCATGG CGGCCGACGC
1261 GCTGGGCTAC GTCTTGCTGG CGTTCGCGAC GCGAGGCTGG ATGGCCTTCC CCATTATGAT
1321 TCTTCTCGCT TCCGGCGGCA TCGGGATGCC CGCGTTGCAG GCCATGCTGT CCAGGCAGGT
1381 AGATGACGAC CATCAGGGAC AGCTTCAAGG ATCGCTCGCG GCTCTTACCA GCCTAACTTC
1441 GATCACTGGA CCGCTGATCG TCACGGCGAT TTATGCCGCC TCGGCGAGCA CATGGAACGG
1501 GTTGGCATGG ATTGTAGGCG CCGCCCTATA CCTTGTCTGC CTCCCCGCGT TGCGTCGCGG
1561 TGCATGGAGC CGGGCCACCT CGACCTGAAT GGAAGCCGGC GGCACCTCGC TAACGGATTC
1621 ACCACTCCAA GAATTGGAGC CAATCAATTC TTGCGGAGAA CTGTGAATGC GCAAACCAAC
1681 CCTTGGCAGA ACATATCCAT CGCGTCCGCC ATCTCCAGCA GCCGCACGCG GCGCATCTCG
1741 GGCAGCGTTG GGTCCTGGCC ACGGGTGCGC ATGATCGTGC TCCTGTCGTT GAGGACCCGG
1801 CTAGGCTGGC GGGGTTGCCT TACTGGTTAG CAGAATGAAT CACCGATACG CGAGCGAACG
1861 TGAAGCGACT GCTGCTGCAA AACGTCTGCG ACCTGAGCAA CAACATGAAT GGTCTTCGGT
1921 TTCCGTGTTT CGTAAAGTCT GGAAACGCGG AAGTCAGCGC CCTGCACCAT TATGTTCCGG
1981 ATCTGCATCG CAGGATGCTG CTGGCTACCC TGTGGAACAC CTACATCTGT ATTAACGAAG
2041 CGCTGGCATT GACCCTGAGT GATTTTTCTC TGGTCCCGCC GCATCCATAC CGCCAGTTGT
2101 TTACCCTCAC AACGTTCCAG TAACCGGGCA TGTTCATCAT CAGTAACCCG TATCGTGAGC
2161 ATCCTCTCTC GTTTCATCGG TATCATTACC CCCATGAACA GAAATCCCCC TTACACGGAG
2221 GCATCAGTGA CCAAACAGGA AAAAACCGCC CTTAACATGG CCCGCTTTAT CAGAAGCCAG
2281 ACATTAACGC TTCTGGAGAA ACTCAACGAG CTGGACGCGG ATGAACAGGC AGACATCTGT
2341 GAATCGCTTC ACGACCACGC TGATGAGCTT TACCGCAGCT GCCTCGCGCG TTTCGGTGAT
2401 GACGGTGAAA ACCTCTGACA CATGCAGCTC CCGGAGACGG TCACAGCTTG TCTGTAAGCG
2461 GATGCCGGGA GCAGACAAGC CCGTCAGGGC GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC
2521 GCAGCCATGA CCCAGTCACG TAGCGATAGC GGAGTGTATA CTGGCTTAAC TATGCGGCAT
2581 CAGAGCAGAT TGTACTGAGA GTGCACCATA TATGCGGTGT GAAATACCGC ACAGATGCGT
2641 AAGGAGAAAA TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC
2701 GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC
2761 AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA
2821 CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA
2881 CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC
2941 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA
3001 CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA
3061 TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA
3121 GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA
3181 CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG
3241 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA CAGTATTTGG
3301 TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG
3361 CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG
3421 AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA
3481 CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT
3541 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC
3601 TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC
3661 ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC
3721 TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC
3781 AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC
3841 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT
3901 GCGCAACGTT GTTGCCATTG CTGCAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC
3961 TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA
4021 AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT
4081 ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG
4141 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC
4201 GAGTTGCTCT TGCCCGGCGT CAACACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA
4261 AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT
4321 GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT
4381 CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG
4441 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA
4501 TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT
4561 AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT
4621 CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTTC AAGAA
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加热灭活酶。通过凝胶提取试剂盒纯化消化的pET-32α（+）载体骨架和从pMD18-T载体切下的基因片段。用T4 DNA连接酶在23℃下用pET-32α（+）载体骨架连接基因片段1小时，载体末端和插入末端的比例为1：3（fmol）。将10μl连接反应与100μl感受态大肠杆菌 BL21（DE3）在冰上混合10分钟，并将混合物在42℃下孵育90秒。加入800μlLB并在37°C下孵育40分钟。涂在含有100μg/ ml氨苄青霉素的LB平板上。在37°C孵育过夜。通过菌落PCR筛选插入物的存在或制备
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相关专题 1)噬菌体DE3溶源化的菌株如BL21(DE3)是最常用的表达菌株，噬菌体DE3是λ噬菌体的衍生株，构建好的表达载体 可以直接转入表达菌株中，诱导蛋白的表达方式与lac启动子一样都是IPTG诱导。 2)表达菌株BL21(DE3)上带有lacI基因，T7 RNA 聚合酶编码基因以及lacUV5启动子，lacUV5启动子可以启动T7 RNA 聚合酶的表达; 3)表达质粒 如pET-28质粒带有T7启动子和编码阻遏
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pET-12c(+)载体
¥1100
