pET-30b
PVT0085 2ug
pET-30b Information
Promoter: T7/lac
Replicon: ColE1 ori, F1 ori
Terminator: T7 Terminator
Plasmid classification: Escherichia coli vector; PET series expression plasmid
Plasmid size: 5421bp
Plasmid tagging: N-6 * His, N-S, N-Thrombin, N-EK, C-6 * H
Prokaryotic resistance: kanamycin Kan
Clonal strain: DH5 alpha
Culture conditions: 37 C, aerobic, LB
Expression host: BL21 (DE3)
Culture conditions: 37 C, aerobic, LB
Induction: IPTG or lactose and its analogues.
5'sequencing primers: T7:TAATACGACTCACTATAGGG
3'sequencing primers: T7-ter:TGCTAGTTATTGCTCAGCGG
pET-30b Description
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 .
pET-30b Multiple cloning site
pET-30b Sequence
LOCUS Exported 5421 bp ds-DNA circular SYN 05-8-2015
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5421)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-8-5
FEATURES Location/Qualifiers
source 1..5421
/organism="synthetic DNA construct"
/mol_type="other DNA"
source 354..376
/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(218..232)
/codon_start=1
/product="enterokinase recognition and cleavage site"
/note="enterokinase site"
/translation="DDDDK"
CDS complement(248..292)
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/note="S-Tag"
/translation="KETAAAKFERQHMDS"
CDS complement(299..316)
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
CDS complement(326..343)
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
RBS 354..376
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 391..415
/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(416..434)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 747..824
/gene="lacI"
/note="lacI promoter"
CDS 825..1907
/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 2716..2907
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 3009..3151
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(3337..3925)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4047..4862
/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(4955..5410)
/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 tccgatatcg ccatggcctt gtcgtcgtcg tcggtaccca
241 gatctgggct gtccatgtgc tggcgttcga atttagcagc agcggtttct ttcataccag
301 aaccgcgtgg caccagacca gaagaatgat gatgatgatg gtgcatatgt atatctcctt
361 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
421 agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat
481 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
541 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
601 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
661 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
721 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
781 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
841 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
901 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
961 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
1021 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
1081 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
1141 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
1201 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
1261 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
1321 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
1381 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
1441 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
1501 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
1561 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
1621 cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
1681 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
1741 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
1801 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
1861 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
1921 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
1981 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
2041 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
2101 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
2161 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
2221 caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg
2281 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
2341 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
2401 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
2461 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
2521 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
2581 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
2641 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
2701 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
2761 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
2821 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt
2881 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
2941 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
3001 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
3061 atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg aaataccgca
3121 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
3181 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
3241 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
3301 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
3361 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
3421 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
3481 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
3541 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
3601 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
3661 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
3721 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
3781 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
3841 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
3901 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
3961 tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt
4021 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
4081 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
4141 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
4201 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
4261 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
4321 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
4381 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
4441 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
4501 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
4561 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt
4621 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
4681 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
4741 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
4801 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
4861 aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
4921 gttccgcgca catttccccg aaaagtgcca cctgaaattg taaacgttaa tattttgtta
4981 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
5041 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
5101 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
5161 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
5221 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
5281 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
5341 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
5401 cagggcgcgt cccattcgcc a
//
Search name
pET-30b,Plasmid pET-30b,pET-30b vector
