| 质粒名称: | pEVOL-pAzF |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 31186 |
| 存储实验室: | Peter Schultz |
|
载体骨架基本信息 |
|
| 载体名称: | p15A |
| 载体出品公司: | -- |
| 空载体大小: | 5000 bp |
| 完整质粒大小: | 6125 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氯霉素，25μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | M.j. p-azidohenylalanine RS (2 copies +tRNA) |
| 物种来源: | M.jannaschii |
| 插入大小: | 1000 bp |
| 基因突变: | Y32T, E107N, D158P, I159L, L162Q, D286R |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BglII(未破坏) |
| 3’克隆位点: | SalI (未破坏) |
| 5’测序引物 | ATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTT |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pEVOL-pAzF was a gift from Peter Schultz (Addgene plasmid # 31186 ; http://n2t.net/addgene:31186 ; RRID:Addgene_31186) |
| 参考文献部分: | Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG. J Am Chem Soc. 2002 Aug 7;124(31):9026-7. 10.1021/ja027007w PubMed 12148987 |
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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 pEVOL-pAzF 6125 bp ds-DNA circular SYN 11-JUN-2021
DEFINITION tRNA synthetase/tRNA pair for the in vivo incorporation of a
photocrosslinker, p-azido-l-phenylalanine, into proteins in E coli
response to the amber codon, TAG.
ACCESSION .
VERSION .
KEYWORDS pEVOL-pAzF
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6125)
AUTHORS Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG
TITLE Addition of p-azido-L-phenylalanine to the genetic code of
Escherichia coli.
JOURNAL J Am Chem Soc. 2002 Aug 7;124(31):9026-7.
PUBMED 12148987
REFERENCE 2 (bases 1 to 6125)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 11, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6125
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 585..602
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(658..675)
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
CDS complement(2578..3237)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 3152..3171
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(3238..3340)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
primer_bind complement(3732..3749)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3866..4411)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(3946..3965)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4557..5435)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 5462..5746
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
primer_bind 5669..5688
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
ORIGIN
1 cgttgaatgt gtaccgtctg gcgctgaaaa ccacgctgaa acgcgcccgt cgttccatgg
61 agctgattgc gcgcgaggat gagaatccaa aagttgctga ggttatttac cctattatgc
121 aagttaatcc gttgcactac cagggtgttg atgttgccgt cggtggtatg gagcaacgca
181 aaattcacat gctggcacgt gaactgctgc cgaaaaaggt tgtctgtatt cataatccgg
241 tcctgaccgg cctggatggc gagggtaaaa tgagcagcag caagggtaac tttattgcag
301 ttgacgatag cccggaagaa atccgtgcga agatcaagaa agcgtactgc ccggcaggcg
361 tggttgaggg taacccgatc atggaaatcg ccaagtattt tctggaatac ccactgacga
421 ttaagcgccc ggagaaattt ggcggcgacc tgaccgtcaa cagctacgag gagctggaaa
481 gcttgtttaa gaacaaagaa ctgcatccga tgcgcctgaa aaacgccgtg gcggaagagc
541 tgattaagat tctggaacca attcgcaaac gtctgtaagt cgaccatcat catcatcatc
601 attgagttta aacggtctcc agcttggctg ttttggcgga tgagagaaga ttttcagcct
661 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
721 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
781 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
841 aggctcagtc gaaagactgg gccttgtttg tgagctcccg gtcatcaatc atccccataa
901 tccttgttag attatcaatt ttaaaaaact aacagttgtc agcctgtccc gctttaatat
961 catacgccgt tatacgttgt ttacgctttg aggaatccca tatggacgag ttcgaaatga
1021 ttaaacgcaa caccagcgaa attatctctg aagaagagct gcgcgaggtg ctgaagaaag
1081 acgagaagag cgcgactatt ggctttgagc cgtccggtaa aattcacctg ggtcactacc
1141 tgcaaatcaa gaagatgatt gatctgcaaa acgctggttt tgacatcatt atcctgctgg
1201 cggacctgca cgcctacctg aatcaaaagg gcgagctgga tgagattcgc aagatcggcg
1261 actacaataa gaaagtcttc gaagccatgg gtttgaaggc taaatacgtc tacggtagca
1321 attttcagct ggataaggat tacacgttga atgtgtaccg tctggcgctg aaaaccacgc
1381 tgaaacgcgc ccgtcgttcc atggagctga ttgcgcgcga ggatgagaat ccaaaagttg
1441 ctgaggttat ttaccctatt atgcaagtta atccgttgca ctaccagggt gttgatgttg
1501 ccgtcggtgg tatggagcaa cgcaaaattc acatgctggc acgtgaactg ctgccgaaaa
1561 aggttgtctg tattcataat ccggtcctga ccggcctgga tggcgagggt aaaatgagca
1621 gcagcaaggg taactttatt gcagttgacg atagcccgga agaaatccgt gcgaagatca
1681 agaaagcgta ctgcccggca ggcgtggttg agggtaaccc gatcatggaa atcgccaagt
1741 attttctgga atacccactg acgattaagc gcccggagaa atttggcggc gacctgaccg
1801 tcaacagcta cgaggagctg gaaagcttgt ttaagaacaa agaactgcat ccgatgcgcc
1861 tgaaaaacgc cgtggcggaa gagctgatta agattctgga accaattcgc aaacgtctgt
1921 aactgcagtt tcaaacgcta aattgcctga tgcgctacgc ttatcaggcc tacatgatct
1981 ctgcaatata ttgagtttgc gtgcttttgt aggccggata aggcgttcac gccgcatccg
2041 gcaagaaaca gcaaacaatc caaaacgccg cgttcagcgg cgttttttct gcttttcttc
2101 gcgaattaat tccgcttcgc aacatgtgag caccggttta ttgactaccg gaagcagtgt
2161 gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat
2221 aattgacgat atgatcagtg cacggctaac taagcggcct gctgactttc tcgccgatca
2281 aaaggcattt tgctattaag ggattgacga gggcgtatct gcgcagtaag atgcgccccg
2341 cattccggcg gtagttcagc agggcagaac ggcggactct aaatccgcat ggcaggggtt
2401 caaatcccct ccgccggacc aaattcgaaa agcctgctca acgagcaggc ttttttgcat
2461 gctcgagcag ctcagggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc
2521 acttattcag gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta
2581 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
2641 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
2701 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
2761 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
2821 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
2881 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
2941 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
3001 gtctttcatt gccatacgga attccggatg agcattcatc aggcgggcaa gaatgtgaat
3061 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
3121 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
3181 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
3241 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
3301 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa
3361 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
3421 atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact
3481 gatttagtgt atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc
3541 cctcctgttc agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc
3601 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
3661 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
3721 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
3781 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
3841 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
3901 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
3961 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
4021 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
4081 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
4141 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
4201 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
4261 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
4321 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
4381 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
4441 tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
4501 atataagttg taattctcat gtttgacagc ttatcatcga taagcttggt acccaattat
4561 gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg
4621 ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca
4681 ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg
4741 atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt
4801 gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg
4861 tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga
4921 tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc
4981 gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg
5041 tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt
5101 gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga
5161 taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc
5221 aaaatatcac tcggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg
5281 cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg
5341 agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt aaacgagtat
5401 cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc gccattcaga
5461 gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt ttactggctc
5521 ttctcgctaa ccaaaccggt aaccccgctt attaaaagca ttctgtaaca aagcgggacc
5581 aaagccatga caaaaacgcg taacaaaagt gtctataatc acggcagaaa agtccacatt
5641 gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat aagattagcg
5701 gatcctacct gacgcttttt atcgcaactc tctactgttt ctccataccc gtttttttgg
5761 gctaacagga ggaattagat ctatggacga gttcgaaatg attaaacgca acaccagcga
5821 aattatctct gaagaagagc tgcgcgaggt gctgaagaaa gacgagaaga gcgcgactat
5881 tggctttgag ccgtccggta aaattcacct gggtcactac ctgcaaatca agaagatgat
5941 tgatctgcaa aacgctggtt ttgacatcat tatcctgctg gcggacctgc acgcctacct
6001 gaatcaaaag ggcgagctgg atgagattcg caagatcggc gactacaata agaaagtctt
6061 cgaagccatg ggtttgaagg ctaaatacgt ctacggtagc aattttcagc tggataagga
6121 ttaca
//
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