| 质粒名称: | pAzFRS.2.t1 |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 73546 |
| 存储实验室: | Farren Isaacs |
|
载体骨架基本信息 |
|
| 载体名称: | p15A |
| 空载体大小: | 4038 bp |
| 完整质粒大小: | 5000 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | 氯霉素，25 μg/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1: | pAzFRS.2.t1 |
| 插入大小 | 921bp |
| GenBank编号 | KT996140 |
| 标签/融合蛋白: | 合成 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | bglII (unknown if destroyed) |
| 3’克隆位点: | SalI (unknown if destroyed) |
| 5’测序引物 | cataagattagcggatcctacctg |
| 3’测序引物 | CTTCTGCGTTCTGATTTAATCTG |
| 插入基因名称2: | pAzFRS.2.t1 |
| GenBank编号 | KT996140 |
| 标签/融合蛋白: | 合成 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | ndeI (not destroyed) |
| 3’克隆位点: | pstI (not destroyed) |
| 5’测序引物 | CAGATTAAATCAGAACGCAGAAG |
| 3’测序引物 | cgaaagcaaattcgaccct |
|
文献引用方法 |
|
| 材料和方法部分: | pEvol-pAzFRS.2.t1 was a gift from Farren Isaacs (Addgene plasmid # 73546 ; http://n2t.net/addgene:73546 ; RRID:Addgene_73546) |
| 参考文献部分: | Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Amiram M, Haimovich AD, Fan C, Wang YS, Aerni HR, Ntai I, Moonan DW, Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, Soll D, Rinehart J, Isaacs FJ. Nat Biotechnol. 2015 Dec;33(12):1272-1279. doi: 10.1038/nbt.3372. Epub 2015 Nov 16. 10.1038/nbt.3372 PubMed 26571098 |
买家导航
pEvol-pAzFRS.2.t1载体基本信息
pEvol-pAzFRS.2.t1载体图谱质粒图谱和多克隆位点信息
pEvol-pAzFRS.2.t1载体简介
pEvol-pAzFRS.2.t1载体序列
LOCUS pEvol-pAzFRS.2.t1 6128 bp ds-DNA circular SYN 15-JUN-2022
DEFINITION tRNA synthetase/tRNA pair for the in vivo incorporation of several
non-standard amino acids, into proteins in E coli in response to the
amber codon, TAG.
ACCESSION .
VERSION .
KEYWORDS pEvol-pAzFRS.2.t1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6128)
AUTHORS Amiram M, Haimovich AD, Fan C, Wang YS, Aerni HR, Ntai I, Moonan DW,
Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, Soll
D, Rinehart J, Isaacs FJ
TITLE Evolution of translation machinery in recoded bacteria enables
multi-site incorporation of nonstandard amino acids.
JOURNAL Nat Biotechnol. 2015 Dec;33(12):1272-1279. doi: 10.1038/nbt.3372.
Epub 2015 Nov 16.
PUBMED 26571098
REFERENCE 2 (bases 1 to 6128)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 15, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6128
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(556..840)
/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 complement(614..633)
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
CDS 867..1745
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
rep_origin 1891..2436
/direction=RIGHT
/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 2337..2356
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2553..2570
/label=L4440
/note="L4440 vector, forward primer"
promoter 2962..3064
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 3065..3724
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(3131..3150)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
primer_bind 5627..5644
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
CDS complement(5700..5717)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
ORIGIN
1 ctgctccatc cctccaacag caacatcaac gcccctataa tgacaaccat taacctgcat
61 tattggatag ataacttcag caacctttgg attttcatcc tctcttgcta taagttccat
121 actccttctt gctcttttta aggtagtttt taaagccaat ctatagacat tcagtgtata
181 atccttatca agcatataag tacttccata aacatatttt gcctttaacc ccattgcttc
241 aaaaactttt ttgttataat ctcctatttt tctaatctca tccaactctc ctttctggtt
301 taaataggcg tgtaaatcag ccaacaatat aattatatca aatccagcat tttgtaaatc
361 aatcatcttt tttatttgga gataatgccc taaatgtatt ttaccacttg gttcaaaacc
421 tatcagagca gatttttcat ctttttttaa aacctctctt aactcttcct cgctgataat
481 ttcagatgtg tttctcttta tcatttcaaa ttcgtccata gatctaattc ctcctgttag
541 cccaaaaaaa cgggtatgga gaaacagtag agagttgcga taaaaagcgt caggtaggat
601 ccgctaatct tatggataaa aatgctatgg catagcaaag tgtgacgccg tgcaaataat
661 caatgtggac ttttctgccg tgattataga cacttttgtt acgcgttttt gtcatggctt
721 tggtcccgct ttgttacaga atgcttttaa taagcggggt taccggtttg gttagcgaga
781 agagccagta aaagacgcag tgacggcaat gtctgatgca atatggacaa ttggtttctt
841 ctctgaatgg cgggagtatg aaaagtatgg ctgaagcgca aaatgatccc ctgctgccgg
901 gatactcgtt taatgcccat ctggtggcgg gtttaacgcc gattgaggcc aacggttatc
961 tcgatttttt tatcgaccga ccgctgggaa tgaaaggtta tattctcaat ctcaccattc
1021 gcggtcaggg ggtggtgaaa aatcagggac gagaatttgt ttgccgaccg agtgatattt
1081 tgctgttccc gccaggagag attcatcact acggtcgtca tccggaggct cgcgaatggt
1141 atcaccagtg ggtttacttt cgtccgcgcg cctactggca tgaatggctt aactggccgt
1201 caatatttgc caatacgggg ttctttcgcc cggatgaagc gcaccagccg catttcagcg
1261 acctgtttgg gcaaatcatt aacgccgggc aaggggaagg gcgctattcg gagctgctgg
1321 cgataaatct gcttgagcaa ttgttactgc ggcgcatgga agcgattaac gagtcgctcc
1381 atccaccgat ggataatcgg gtacgcgagg cttgtcagta catcagcgat cacctggcag
1441 acagcaattt tgatatcgcc agcgtcgcac agcatgtttg cttgtcgccg tcgcgtctgt
1501 cacatctttt ccgccagcag ttagggatta gcgtcttaag ctggcgcgag gaccaacgta
1561 tcagccaggc gaagctgctt ttgagcacca cccggatgcc tatcgccacc gtcggtcgca
1621 atgttggttt tgacgatcaa ctctatttct cgcgggtatt taaaaaatgc accggggcca
1681 gcccgagcga gttccgtgcc ggttgtgaag aaaaagtgaa tgatgtagcc gtcaagttgt
1741 cataattggg taccaagctt atcgatgata agctgtcaaa catgagaatt acaacttata
1801 tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg aaatctagaa
1861 atattttatc tgattaataa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc
1921 ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac
1981 caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc
2041 agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg
2101 ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg
2161 ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa
2221 ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga
2281 atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga
2341 aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt
2401 tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca
2461 cttccctgtt aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca
2521 tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat
2581 atatcctgta tcacatattc tgctgacgca ccggtgcagc cttttttctc ctgccacatg
2641 aagcacttca ctgacaccct catcagtgcc aacatagtaa gccagtatac actccgctag
2701 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag
2761 ggacagctga tagaaacaga agccactgga gcacctcaaa aacaccatca tacactaaat
2821 cagtaagttg gcagcatcac ccgacgcact ttgcgccgaa taaatacctg tgacggaaga
2881 tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa
2941 cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga
3001 aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc
3061 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
3121 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
3181 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
3241 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga
3301 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
3361 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
3421 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
3481 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
3541 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca
3601 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
3661 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
3721 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaataag
3781 tgataataag cggatgaatg gcagaaattc gaaagcaaat tcgaccctga gctgctcgag
3841 catgcaaaaa agcctgctcg ttgagcaggc ttttcgaatt tggtccggcg gaggggattt
3901 gaacccctgc catgcggatt tagagtccgc cgttctgccc tgctgaacta ccgccggaat
3961 gcggggcgca tcttactgcg cagatacgcc ctcgtcaatc ccttaatagc aaaatgcctt
4021 ttgatcggcg agaaagtcag caggccgctt agttagccgt gcactgatca tatcgtcaat
4081 tattacctcc acggggagag cctgagcaaa ctggcctcag gcatttgaga agcacacggt
4141 cacactgctt ccggtagtca ataaaccggt gctcacatgt tgcgaagcgg aattaattcg
4201 cgaagaaaag cagaaaaaac gccgctgaac gcggcgtttt ggattgtttg ctgtttcttg
4261 ccggatgcgg cgtgaacgcc ttatccggcc tacaaaagca cgcaaactca atatattgca
4321 gagatcatgt aggcctgata agcgtagcgc atcaggcaat ttagcgtttg aaactgcagt
4381 tataatctct ttctaattgg ctctaaaatc tttataagtt cttcagctac agcatttttt
4441 aagcgcattg gatgcaattc cttattttta aataaactct ctaactcctc atagctatta
4501 actgtcaaat ctccaccaaa tttttctggc ccttttatgg ttaaaggata ttcaaggaag
4561 tatttagcta tctccattat tggatttcct tcaacaactc cagctgggca gtatgctttc
4621 tttatcttag ccctaatctc ttctggagag tcatcaacag ctataaaatt cccttttgaa
4681 gaactcatct ttccttctcc atccaaaccc gttaagacag ggttgtgaat acaaacaacc
4741 ttttttggta aaagctccct tgctaacatg tgtatttttc tctgctccat ccctccaaca
4801 gcaacatcaa cgcccctata atgacaacca ttaacctgca ttattggata gataacttca
4861 gcaacctttg gattttcatc ctctcttgct ataagttcca tactccttct tgcccttttt
4921 aaggtagttt ttaaagccaa tctatagaca ttcagtgtat aatccttatc aagcatataa
4981 gtacttccat aaacatattt tgcctttaac cccattgctt caaaaacttt tttgttataa
5041 tctcctattt ttctaatctc atccaactct cctttctggt ttaaataggc gtgtaaatca
5101 gccaacaata taattatatc aaatccagca ttttgtaaat caatcatctt ttttatttgg
5161 agataatgcc ctaaatgtat tttaccactt ggttcaaaac ctatcagagc agatttttca
5221 tcctttttta aaacctctct taactcttcc tcgctgataa tttcagatgt gtttctcttt
5281 atcatttcaa attcgtccat atgggattcc tcaaagcgta aacaacgtat aacggcgtat
5341 gatattaaag cgggacaggc tgacaactgt tagtttttta aaattgataa tctaacaagg
5401 attatgggga tgattgatga ccgggagctc acaaacaagg cccagtcttt cgactgagcc
5461 tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagacc ccacactacc
5521 atcggcgcta cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct
5581 actgccgcca ggcaaattct gttttatcag accgcttctg cgttctgatt taatctgtat
5641 caggctgaaa atcttctctc atccgccaaa acagccaagc tggagaccgt ttaaactcaa
5701 tgatgatgat gatgatggtc gacttattat aatctctttc taattggctc taaaatcttt
5761 ataagttctt cagctacagc attttttaag cgcattggat gcaattcctt atttttaaat
5821 aaactctcta actcctcata gctattaact gtcaaatctc caccaaattt ttctggccct
5881 tttatggtta aaggatattc aaggaagtat ttagctatct ccattattgg atttccttca
5941 acaactccag ctgggcagta tgctttcttt atcttagccc taatctcttc tggagagtca
6001 tcaacagcta taaaattccc ttttgaagaa ctcatctttc cttctccatc caaacccgtt
6061 aagacagggt tgtgaataca aacaaccttt tttggtaaaa gctcccttgc taacatgtgt
6121 atttttct
//
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