| 质粒名称: | pSecUAG-Evol2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 163148 |
| 存储实验室: | Dieter Söll |
|
载体骨架基本信息 |
|
| 载体名称: | pBAD-myc-HisA |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 3500 bp |
| 完整质粒大小: | 8635 bp |
| 载体修饰: | RSF来源和卡那霉素耐药性取自pRSF-Duet1，并用于取代pBAD-myc-HisA的pBR322来源和氨苄青霉素耐药性 |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 卡那霉素，50μg/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: | allo-tRNA UTu2D |
| 物种来源1: | Synthetic |
| 插入大小1: | 90 bp |
| 启动子1: | araBAD |
| 标签/融合蛋白1: | -- |
| 克隆方法1: | Gibson Cloning |
| 5’测序引物1 | ATGCCATAGCATTTTTATCC |
| 3’测序引物1 | -- |
| 插入基因名称2: | 硫氧还蛋白 |
| 物种来源2: | Treponema denticola |
| 插入大小2: | 324 bp |
| 基因突变2: | Cysteine 32 switched to Selenocysteine (UAG) |
| GenBank编号2 | NC_002967 |
| 克隆方法2: | Gibson Cloning |
| 5’测序引物2 | GCGGCTATTTAACGACCCTG |
| 插入基因名称3 | Selenophosphate synthase |
| 物种来源3 | 鲑鱼气单胞菌 |
| 插入大小3 | 1038 bp |
| 基因突变3 | starts with GUG instead of AUG |
| GenBank编号3 | NF_002098 |
| 启动子3 | native As SelD promoter |
| 克隆方法3 | Gibson Cloning |
| 5’测序引物3 | CTACCACGGTGCCCAAAAGTC |
| 3’测序引物3 | GCATTTTCGACAGGCTATTG |
| 插入基因名称4 | 硒半胱氨酸合成酶 |
| 物种来源4 | 鲑鱼气单胞菌 |
| 插入大小4 | 1488 bp |
| 基因突变4 | 将引发剂AUG改为GUG，然后添加以下氨基酸P2T、L69F、Q72S和C173V |
| GenBank编号4 | NF_003308 |
| 启动子4 | EM7 |
| 克隆方法4 | Gibson Cloning |
| 5’测序引物4 | CAGGGCATTATGCCACAAG |
| 3’测序引物4 | GAAGTGGATCGTCTGGTGAC |
| 插入基因名称5 | 半胱氨酸脱硫酶 |
| 物种来源5 | 大肠杆菌 |
| 插入大小5 | 1221 bp |
| 基因突变5 | 半胱氨酸364至丙氨酸 |
| GenBank编号5 | NF_006791 |
| 启动子5 | native Ec SufS promoter |
| 克隆方法5 | Gibson Cloning |
| 5’测序引物5 | GATTCGGGCTGACTGTGAGAG |
| 3’测序引物5 | CTCGGTGAGTTTTCTCCTTC |
|
文献引用方法 |
|
| 材料和方法部分: | pSecUAG-Evol2 was a gift from Dieter Söll (Addgene plasmid # 163148 ; http://n2t.net/addgene:163148 ; RRID:Addgene_163148) |
| 参考文献部分: | A Facile Method for Producing Selenocysteine-Containing Proteins. Mukai T, Sevostyanova A, Suzuki T, Fu X, Soll D. Angew Chem Int Ed Engl. 2018 Jun 11;57(24):7215-7219. doi: 10.1002/anie.201713215. Epub 2018 May 9. 10.1002/anie.201713215 PubMed 29631320 |
买家导航
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 pSecUAG-Evol2 8649 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses machinery necessary for selenocysteine incorporation at
UAG codons.
ACCESSION .
VERSION .
KEYWORDS pSecUAG-Evol2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8649)
AUTHORS Mukai T, Sevostyanova A, Suzuki T, Fu X, Soll D
TITLE A Facile Method for Producing Selenocysteine-Containing Proteins.
JOURNAL Angew Chem Int Ed Engl. 2018 Jun 11;57(24):7215-7219. doi:
10.1002/anie.201713215. Epub 2018 May 9.
PUBMED 29631320
REFERENCE 2 (bases 1 to 8649)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8649
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(1..285)
/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(59..78)
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
CDS 312..1190
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
primer_bind complement(1346..1368)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 1454..1594
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 1509..1528
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2294..2313
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 2579..2670
/gene="bla"
/label=AmpR promoter
CDS 2671..3486
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(2744..2763)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
promoter 4741..4788
/label=EM7 promoter
/note="synthetic bacterial promoter "
terminator 8214..8300
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 8453..8470
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
ORIGIN
1 atggagaaac agtagagagt tgcgataaaa agcgtcaggt aggatccgct aatcttatgg
61 ataaaaatgc tatggcatag caaagtgtga cgccgtgcaa ataatcaatg tggacttttc
121 tgccgtgatt atagacactt ttgttacgcg tttttgtcat ggctttggtc ccgctttgtt
181 acagaatgct tttaataagc ggggttaccg gtttggttag cgagaagagc cagtaaaaga
241 cgcagtgacg gcaatgtctg atgcaatatg gacaattggt ttcttctctg aatggcggga
301 gtatgaaaag tatggctgaa gcgcaaaatg atcccctgct gccgggatac tcgtttaatg
361 cccatctggt ggcgggttta acgccgattg aggccaacgg ttatctcgat ttttttatcg
421 accgaccgct gggaatgaaa ggttatattc tcaatctcac cattcgcggt cagggggtgg
481 tgaaaaatca gggacgagaa tttgtttgcc gaccgggtga tattttgctg ttcccgccag
541 gagagattca tcactacggt cgtcatccgg aggctcgcga atggtatcac cagtgggttt
601 actttcgtcc gcgcgcctac tggcatgaat ggcttaactg gccgtcaata tttgccaata
661 cggggttctt tcgcccggat gaagcgcacc agccgcattt cagcgacctg tttgggcaaa
721 tcattaacgc cgggcaaggg gaagggcgct attcggagct gctggcgata aatctgcttg
781 agcaattgtt actgcggcgc atggaagcga ttaacgagtc gctccatcca ccgatggata
841 atcgggtacg cgaggcttgt cagtacatca gcgatcacct ggcagacagc aattttgata
901 tcgccagcgt cgcacagcat gtttgcttgt cgccgtcgcg tctgtcacat cttttccgcc
961 agcagttagg gattagcgtc ttaagctggc gcgaggacca acgtatcagc caggcgaagc
1021 tgcttttgag caccacccgg atgcctatcg ccaccgtcgg tcgcaatgtt ggttttgacg
1081 atcaactcta tttctcgcgg gtatttaaaa aatgcaccgg ggccagcccg agcgagttcc
1141 gtgccggttg tgaagaaaaa gtgaatgatg tagccgtcaa gttgtcataa ttggtaacga
1201 atcagacaat tgacggcttg acggagtagc atagggtttg cagaatccct gcttcgtcca
1261 tttgacaggc acattatgca tgccgcttcg ccttcgcgcg cgaattgatc tgctgcctcg
1321 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
1381 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
1441 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
1501 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
1561 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
1621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
1681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
1741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccggccggc
1801 cgaaatcggc cggcccggaa tagctgttcg ttgacttgat agaccgattg attcatcatc
1861 tcataaataa agaaaaacca ccgctaccaa cggtggtttt ctcaaggttc gctgagctac
1921 caactctttg aaccaaggta agtgggttgg aggaccgcac tcaccaaaat ctgttctttc
1981 agtttagcct taacaggtgc ataacttcaa gacaaagtcc tctaaatcag ttaccaatgg
2041 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
2101 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
2161 cgacctacac cgaactgaga taccaacagc gtgagctatg agaaagcgcc acgcttcccg
2221 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
2281 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
2341 ggcttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcct
2401 gcggcgttgg cttcttccgg tgctttgctt tttgctcaca tgttctttcc ggctttatcc
2461 cctgattctg tggataaccg tattaccgct tttgagtgag ctgacaccgc tcgccgcagt
2521 cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aagagcgctg catgcctatt
2581 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
2641 atgcttcaat aatattgaaa aaggaagagt atgagccata ttcaacggga aacgtcttgc
2701 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
2761 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
2821 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
2881 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
2941 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
3001 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
3061 ttgcattcga ttcctgtttg taattgtcct tttaacagcg accgcgtatt tcgtctcgct
3121 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
3181 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
3241 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
3301 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
3361 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
3421 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
3481 ttctaagaat taataactgc caggaggtgc aagatgattt tttccgtcga caaagtgcgg
3541 gccgactttc cggtgctttc gcgtgaggta aacggtttgc cgctggctta tctcgacagc
3601 gccgccagtg cgcagaaacc gagccaggtg attgacgccg aggccgagtt ttatcgtcat
3661 ggctacgcgg cggtgcatcg tggtattcat accttaagcg cccaggcgac cgagaaaatg
3721 gagaacgtgc gcaagcgggc atcgctgttt attaatgccc gttcggcgga agagctggtg
3781 ttcgtccgcg gcacgacgga agggatcaat ctggtcgcca atagctgggg caacagcaac
3841 gtgcgggcgg gcgataacat catcatcagt cagatggagc accacgctaa cattgttccc
3901 tggcagatgc tttgcgcacg cgttggcgca gagctgcgtg tgatcccgct caatcccgat
3961 ggtacgttgc aactggagac gctgcctacg ctgtttgatg agaaaactcg cctgctggca
4021 attactcatg tctccaacgt gcttggcaca gaaaatccac tggcggaaat gatcacgctt
4081 gcgcaccagc atggcgcaaa agtgctggtg gatggcgctc aggcggtgat gcatcatccg
4141 gtggatgttc aggcgctgga ttgcgacttt tacgtgttct ccgggcataa actgtatggc
4201 cccaccggaa ttggcattct ttatgtgaaa gaagccttgt tgcaggagat gccgccgtgg
4261 gaagggggcg gttctatgat cgccaccgtc agcctgagtg aaggcactac ctggaccaaa
4321 gcaccatggc ggtttgaagc cggtacaccc aataccgggg gcatcattgg tcttggcgcg
4381 gcgctggagt atgtttcggc gctggggctt aataacatag ccgagtatga acagaatctg
4441 atgcattatg cgctatcaca gctggaatct gtaccggatc tcactctcta tggcccacaa
4501 aacaggcttg gcgttattgc ttttaatctc ggtaaacacc acgcctatga tgttggcagt
4561 tttctcgata attacggcat tgctgtgcgt accggacatc acgccgcaat gccattgatg
4621 gcctattaca acgtccctgc gatgtgtcgg gcgtcgctgg ccatgtataa cacccatgaa
4681 gaagtggatc gtctggtgac cggcctgcaa cgtattcacc gtttgctggg ataatcacgt
4741 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
4801 taaaccgtga cgaactcgtc tcacgcgcca gccatcgcca gttctgctag tcagcccgaa
4861 tcatgtccca ctgccgacga ttcactgcca gattcactgc cagattcact gccacagccc
4921 agccagcaac aagcgcgccg tctaccgcaa gtggaacagc tgctgcagca accctttctc
4981 accggtttta tcgaggcgct gagccgccct tttgtgacct cggcggtgcg cgatgtcctg
5041 agcgaattgc gccagagcga ggcatttcgc cagcatgggg ttgcccccga gcaaatcgag
5101 gcactgattg ccaagcgttg ccagcagcag ctgcgccaac gtcagacccg ggtgatcaac
5161 gccaccggca ccctggtgca caccaatctg gggcgctcgc cgctaagtcg cgagctgtgg
5221 gacgaggtgc gcgacctcaa cactggctac aacaatctgg aactggatct cgccaccggc
5281 aagcgcggcg ggcgcaaggg gctgatcgcc cccctgctcc gtgtgctcac ccaggccgag
5341 gattcgctgg tggtcaacaa caacgccgct tcgctcttct tgctgctgca ggagatagcc
5401 aaggggcgcg aggtgatcgt ctcgcggggc gaacagatcc agattggtgg cggctttcgc
5461 attcccgaca ttctggcgct ctccggcgcc aaactggtgg aggtgggcac caccaatatc
5521 actaccgcca aagattacct cgatgccatc acagatcaga ccgcgctggt gctgatggta
5581 cacagatcca atttcgccat tcgcggcttt accgaatccc ccgatattgg cgaggtggcc
5641 cgcgccctgc ccgagcacgt ggtgctggcg gtggatcagg gctcgggctt gaccaccgag
5701 gagtttgcac cggacgaaac ctcggtgcgt cagtacatca aggcgggggc ggatctggtc
5761 tgctactccg gcgacaagct gctgggtggc ccgcaatcgg gcatcatcag cggccgcagc
5821 gacctcatca agcggctgga aaaacacccc atgatgcgca ccttccgccc gagccgcatc
5881 gtctactccc tgctggaacg cctgctcatc cacaagctca acaagtcccc catcggcgag
5941 ggcatcgccc agcgcacctt gagcaaccct gccgccatgc aggcccgcgc cgatcagctg
6001 atggccgccc tgcccggctg ctttgtgccg gtccccgccc agctggtggt gggtggtggc
6061 accctgccgg acgagttcta ccctgcgcct gcgctcgaat gcaccgaccc gcgtccggcc
6121 cagcagctgc tcgatgccct gcggaaactg ccggtgccgg tcatcgccac cgtgcgccag
6181 cagaaggtgc tgctcaatat ggcgaccctg ctgccgaccg agattgcact gcttatcgcc
6241 caactcaagg agttgctact gcccactccg accactgcga ccgaggagcc ctgatcccac
6301 agccgccagt tccgctggcg gcattttcga caggctattg tgattcgaac ccgcagagtc
6361 cttgtggcat aatgccctgt ccaatggccc gctcagggtc atctcctttt gcctgatata
6421 cgctgtttca tcttgagaaa cagccaaaca aggggtaatc gtgtcttcca ttcgtctgac
6481 ccaatacagc cacggggctg gctgcggctg caaaatttct cccaaggtgc tcgacaccat
6541 tctcaagagc cagatcccgg gctttgacga cccgaccctg gtggttggca acagcagcaa
6601 ggatgacgcg gccgtggtcg atatcggcaa cggtcagggc attgtttcca ccaccgactt
6661 cttcatgccc atcgtcgatg atccctttac ctttggccgc atcgcggcca ccaacgccat
6721 cagcgacatc tacgccatgg gcggcaagcc catcgttgcc attgccatcc ttggctggcc
6781 catcaacacc ctagccccgg aagtggccca gcaggtgata gatggcggcc gccaggtgtg
6841 ccatgaagcg ggcatatcct tggctggcgg ccacagtatc gatgcccccg agcccatctt
6901 cggtcttgct gtgaccggta tagtgccgct caatgccatc aagcagaacg acacggccca
6961 ggcgggtgac atcctctacc tgaccaagcc cctcggtatc ggcatcctca ccacggccca
7021 gaagaagggc aaattgaagc cagagcatga gcagctggcc cccaacgcca tgtgcaccct
7081 caacaagatt ggccagcgct ttgccgaact gcccggcgtg cacgccatga cggatgtgac
7141 cgggtttggc ctggcgggac acctgcttga gatgtgcgaa ggctcagggg tgtgtgccac
7201 cctcgatttc aaggcgctgc cactgctcga cgaagtagat tactacctgt ccgagggctg
7261 cgtaccgggc ggtaccctgc gcaacttcga ttcctatggc gccaagctcg gtgccatgga
7321 tgaacgcacc cgcaacatca tgtgcgatcc gcagaccagc ggcggcttgc tggttgccgt
7381 cggtaaagaa agtgaagccg agctccttgc tatcgcgaca caagcggggc tgaccctctc
7441 ccccataggc cagctgaaag cctataccgg aaaccagttt atcgaggtta tccaatgatc
7501 acacaggaaa cagaccatga ttatggcagt attggatatt acaaatgcta attttgatga
7561 aaccgttaag accgccaagc ccgttttaat tgacttttgg gcaccgtggt agccgggatg
7621 cgtacagctc agtcctgagc tgcaggctgc cgaggcggaa ctcggcgaca aggctgtgat
7681 agcacagtct aacgtggata atgcacgtga attggcagta aaatttaagt ttatgtcaat
7741 acctaccctc atcgttttaa aagacggaaa agaggtggac aggcacacag gctatatgga
7801 taagaagagc cttgtaaact ttgtttcaaa gcatatctaa ttcatgagcg gatacatatt
7861 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
7921 acttgcggag acccggtcgt cagcttgtcg tcggttcagg gcggggtcgt taaatagccg
7981 cttatgtcta ttgctggttt accggtttat tgactaccgg aagcagtgtg accgtgtgct
8041 tctcaaatgc ctgaggtttc agcaaaaaac ccctcaagac ccgtttagag gcggccgcgg
8101 ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct
8161 gcccgccacc ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc
8221 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
8281 tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac
8341 actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac
8401 cgcgctactg ccgccaggca aattctgttt tatcagaccg cttctgcgtt ctgatttaat
8461 ctgtatcagg ctgctcggat ccatggttaa ttcctaaaaa aaatccttag ctttcgctaa
8521 ggatctgcag gtcgactcta gatcctggcg gacgggggag gaatcgaacc taacgcctgc
8581 acagcgtcct gacgggttta gagcccgcga ccgccaccag acggaacccc cgtccgaatt
8641 caaacgggt
//
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