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- 规格：20μl质粒
- 启动子：T7/lac
- 复制子：ColE1 ori，F1 ori
- 终止子：T7 terminator
- 质粒分类：大肠杆菌载体；PET系列表达质粒
- 质粒大小：5423bp
- 质粒标签：N-6×His, N-S, N-Thrombin, N-EK, C-6×His
- 原核抗性：卡那霉素Kan
- 克隆菌株：DH5α
- 表达宿主：BL21(DE3)
- 诱导方式：IPTG或乳糖及其类似物
- 5测序引物：T7:TAATACGACTCACTATAGGG
- 3测序引物：T7-ter:TGCTAGTTATTGCTCAGCGG
- 质粒简介： The pET-30a-c(+) vectors carry an N-terminal His• Tag®/ thrombin/ S• Tag™/ enterokinase configuration plus an optional C-terminal His• Tag sequence. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer .
- 质粒图谱1： png/3-161012134043442.png
质粒序列： 点击查看序列LOCUS Exported 5423 bp ds-DNA circular SYN 05-8-2015
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5423)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-8-5 from MLCC
http://www.fenghbio.cn
FEATURES Location/Qualifiers
source 1..5423
/organism="synthetic DNA construct"
/mol_type="other DNA"
source 356..378
/organism="Enterobacteria phage T7"
/mol_type="genomic DNA"
/db_xref="taxon:10760"
terminator 26..73
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS complement(220..234)
/codon_start=1
/product="enterokinase recognition and cleavage site"
/note="enterokinase site"
/translation="DDDDK"
CDS complement(250..294)
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/note="S-Tag"
/translation="KETAAAKFERQHMDS"
CDS complement(301..318)
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
CDS complement(328..345)
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
RBS 356..378
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 393..417
/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)."
promoter complement(418..436)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 749..826
/gene="lacI"
/note="lacI promoter"
CDS 827..1909
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
CDS 2718..2909
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 3011..3153
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(3339..3927)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4049..4864
/codon_start=1
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin complement(4957..5412)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
ORIGIN
1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
181 cgacggagct cgaattcgga tccacagata tcccatggcc ttgtcgtcgt cgtcggtacc
241 cagatctggg ctgtccatgt gctggcgttc gaatttagca gcagcggttt ctttcatacc
301 agaaccgcgt ggcaccagac cagaagaatg atgatgatga tggtgcatat gtatatctcc
361 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
421 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
481 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
541 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
601 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
661 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
721 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
781 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
841 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
901 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
961 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
1021 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
1081 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
1141 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
1201 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
1261 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
1321 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
1381 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
1441 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
1501 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
1561 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
1621 tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
1681 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
1741 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
1801 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
1861 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
1921 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
1981 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
2041 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
2101 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
2161 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
2221 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
2281 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
2341 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
2401 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
2461 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
2521 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
2581 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
2641 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
2701 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
2761 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
2821 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
2881 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
2941 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
3001 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
3061 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg
3121 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
3181 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
3241 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
3301 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
3361 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
3421 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
3481 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
3541 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
3601 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
3661 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
3721 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
3781 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
3841 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
3901 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
3961 gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc
4021 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
4081 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
4141 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
4201 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
4261 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
4321 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
4381 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
4441 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
4501 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
4561 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
4621 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
4681 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
4741 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
4801 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
4861 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
4921 gggttccgcg cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt
4981 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
5041 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
5101 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
5161 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
5221 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
5281 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
5341 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
5401 tacagggcgc gtcccattcg cca
//
