| 质粒名称: | pET22b RNase A |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 58903 |
| 存储实验室: | Ronald Raines |
|
载体骨架基本信息 |
|
| 载体名称: | pET22b(+) |
| 载体出品公司: | EMD Millipore |
| 空载体大小: | 5493 bp |
| 载体类型: | 细菌细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | RNase A |
| 物种来源: | B. taurus (bovine) |
| 插入大小: | 500bp |
| 基因突变: | RNASE1 (a.k.a. RNS1) |
| GenBank编号 | NP_001014408 |
| 启动子: | T7 |
| 标签/融合蛋白: | pelB sequence (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NcoI(未破坏) |
| 3’克隆位点: | SalI (未破坏) |
| 5’测序引物 | T7 |
| 3’测序引物 | T7-Term |
|
文献引用方法 |
|
| 材料和方法部分: | pET22b RNase A was a gift from Ronald Raines (Addgene plasmid # 58903 ; http://n2t.net/addgene:58903 ; RRID:Addgene_58903) |
| 参考文献部分: | Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11. delCardayre SB, Ribo M, Yokel EM, Quirk DJ, Rutter WJ, Raines RT. Protein Eng. 1995 Mar;8(3):261-73. PubMed 7479688 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pET22b RNase A 5828 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Bacterial expression of bovine pancreatic ribonuclease A.
ACCESSION .
VERSION .
KEYWORDS pET22b RNase A
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5828)
AUTHORS delCardayre SB, Ribo M, Yokel EM, Quirk DJ, Rutter WJ, Raines RT
TITLE Engineering ribonuclease A: production, purification and
characterization of wild-type enzyme and mutants at Gln11.
JOURNAL Protein Eng. 1995 Mar;8(3):261-73.
PUBMED 7479688
REFERENCE 2 (bases 1 to 5828)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5828
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 7..26
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 110..129
/label=T7
/note="T7 promoter, forward primer"
promoter 110..128
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 129..153
/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)."
RBS 168..190
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
sig_peptide 198..263
/gene="pelB (fragment)"
/product="leader peptide for secretion"
/label=pelB signal sequence
CDS 264..308
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
CDS 668..685
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(738..756)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 752..799
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 836..1291
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(923..942)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 1133..1154
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 1317..1421
/gene="bla"
/label=AmpR promoter
CDS 1422..2282
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(1640..1659)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2453..3041
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2942..2961
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3195..3212
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 3227..3366
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(3292..3311)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3452..3474
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(3468..3659)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind 5513..5532
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(5552..5629)
/gene="lacI"
/label=lacI promoter
ORIGIN
1 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
61 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
121 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
181 aagaaggaga tatacatatg aaatacctgc tgccgaccgc tgctgctggt ctgctgctcc
241 tcgctgccca gccggcgatg gccaaggaaa ctgcagcagc caagtttgag cggcagcaca
301 tggactccag cacttccgct gccagcagct ccaactactg taaccagatg atgaagagcc
361 ggaacctgac caaagatcga tgcaagccag tgaacacctt tgtgcacgag tccctggctg
421 atgtccaggc cgtgtgctcc cagaaaaatg ttgcctgcaa gaatgggcag accaattgct
481 accagagcta ctccaccatg agcatcaccg actgccgtga gaccggcagc tccaagtacc
541 ccaactgtgc ctacaagacc acccaggcga ataaacacat cattgtggct tgtgagggaa
601 acccgtacgt gccagtccac tttgatgctt cagtgtagta gtcgacaagc ttgcggccgc
661 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
721 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
781 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
841 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
901 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
961 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
1021 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
1081 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
1141 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
1201 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
1261 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
1321 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
1381 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
1441 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
1501 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
1561 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
1621 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
1681 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
1741 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
1801 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
1861 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
1921 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gccagctgca atggcaacaa
1981 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
2041 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
2101 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
2161 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
2221 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
2281 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
2341 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
2401 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
2461 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
2521 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
2581 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
2641 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
2701 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
2761 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
2821 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
2881 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
2941 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
3001 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
3061 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
3121 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
3181 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
3241 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
3301 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
3361 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
3421 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
3481 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
3541 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
3601 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
3661 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
3721 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
3781 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
3841 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
3901 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
3961 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
4021 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
4081 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
4141 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
4201 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
4261 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
4321 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
4381 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
4441 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
4501 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
4561 cagggtggtt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
4621 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
4681 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
4741 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
4801 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
4861 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
4921 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
4981 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
5041 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
5101 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
5161 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
5221 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
5281 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
5341 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
5401 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
5461 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
5521 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
5581 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
5641 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
5701 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
5761 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
5821 cgatcttc
//
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