| 质粒名称: | circRNA-synIRES-R25-NanoLuc |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 188116 |
| 存储实验室: | Howard Chang |
|
载体骨架基本信息 |
|
| 载体名称: | pRC |
| 空载体大小: | 6500bp |
| 完整质粒大小: | 7103 bp |
| 载体类型: | 哺乳细胞表达、Luciferase |
| 载体抗性: | 氨苄霉素 |
| 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: | NanoLuc |
| 物种来源: | Synthetic |
| 插入大小: | 603 bp |
| 启动子: | T7 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI(未破坏) |
| 3’克隆位点: | MfeI (未破坏) |
| 5’测序引物 | GGGACGGGACCGACTACTTTGG |
| 3’测序引物 | CTAGTAGACAATCCCGTGCTAA |
|
文献引用方法 |
|
| 材料和方法部分: | circRNA-synIRES-R25-NanoLuc was a gift from Howard Chang (Addgene plasmid # 188116 ; http://n2t.net/addgene:188116 ; RRID:Addgene_188116) |
| 参考文献部分: | Engineering circular RNA for enhanced protein production. Chen R, Wang SK, Belk JA, Amaya L, Li Z, Cardenas A, Abe BT, Chen CK, Wender PA, Chang HY. Nat Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi: 10.1038/s41587-022-01393-0. 10.1038/s41587-022-01393-0 PubMed 35851375 |
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 circRNA-synIRES-R25-NanoLuc 7103 bp ds-DNA circular SYN 14-OCT-2022
DEFINITION Template plasmid to transcribe circular RNA encoding NanoLuc.
ACCESSION .
VERSION .
KEYWORDS circRNA-synIRES-R25-NanoLuc
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7103)
AUTHORS Chen R, Wang SK, Belk JA, Amaya L, Li Z, Cardenas A, Abe BT, Chen
CK, Wender PA, Chang HY
TITLE Engineering circular RNA for enhanced protein production.
JOURNAL Nat Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi:
10.1038/s41587-022-01393-0.
PUBMED 35851375
REFERENCE 2 (bases 1 to 7103)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 14, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7103
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 158..746
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 647..666
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
enhancer 812..1115
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
intron 1392..2408
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 2332..2354
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 2416..2435
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
primer_bind 2478..2497
/label=T7
/note="T7 promoter, forward primer"
promoter 2478..2496
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 3561..3626
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3642..4154
/codon_start=1
/product="NanoLuc(R) luciferase"
/label=Nluc
/note="human codon-optimized"
/translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
LCERILA"
terminator 4551..4598
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
polyA_signal 4612..4836
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 4879..4895
/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)."
primer_bind 4884..4906
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 4903..4919
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4903..4919
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
polyA_signal 4968..5089
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4979..4998)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 5033..5052
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS complement(5249..5848)
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(5332..5352)
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind 5829..5848
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
promoter complement(5859..6187)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 5873..6008
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind complement(5927..5946)
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS complement(6221..7036)
/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 6944..6963
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 7028..7047
/label=pENTR-R
/note="pENTR vectors, reverse primer"
ORIGIN
1 tgtgcaatgt aacatcagag attttgagac acaacgtggc tttgttgaat aaatcgaact
61 tttgctgagt tgaaggatca gtcatgacca aaatccctta acgtgagttt tcgttccact
121 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
181 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
241 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
301 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
361 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
421 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
481 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
541 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
601 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
661 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
721 cgtcaggggg gcggagccta tggaaacctg caggttatta atagtaatca attacggggt
781 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
841 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
901 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
961 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
1021 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
1081 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
1141 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
1201 tattttgtgc agcgatgggg gcgggggggg ggggggnsgc gccrggsggg gggggggggg
1261 gsgggggggg gggsggggsg rggsggrgag gkgcggcggc agccaatcag agcggcgcgc
1321 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
1381 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
1441 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
1501 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt
1561 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg
1621 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg
1681 gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga
1741 gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg
1801 tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac
1861 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc
1921 gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc
1981 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc
2041 ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt
2101 gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc
2161 gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa
2221 tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc
2281 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
2341 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
2401 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcgg
2461 ccgcggccag tgaattgtaa tacgactcac tataggggtt aacaaaaaaa aaaaaaaaaa
2521 aaaaaaatca ccgaccgatc tatctgaacc ggtgggaatt ctagagaaaa tttcgtctgg
2581 attagttact tatcgtgtaa aatctgataa atggaattgg ttctacataa atgcctaacg
2641 actatccctt tggggagtag ggtcaagtga ctcgaaacga tagacaactt gctttaacaa
2701 gttggagata tagtctgctc tgcatggtga catgcagctg gatataattc cggggtaaga
2761 ttaacgacct tatctgaaca taacgctacc gtttaatatt gcgtcatatt cggcgaccat
2821 ttgtgtggta aaaaaaaaaa accaaaaaaa aaaaacaaaa aaaaaaaata attgactaag
2881 atatcttaaa acagcggatg ggtaccccac catccgaccc actgggtgta gtactctggt
2941 acttcgtacc tttgtacgcc tgttcttccc attgtaccct tcctgaactt ccaacccaag
3001 taacgttaga agctcaacat ttagtacaac aggaagcacc acatccagtg gtgtttagta
3061 caagcacttc tgtttccccg gagcgaggta taggctgtac ccactgccaa aaacctttaa
3121 ccgttatccg ccaaccaact acgtaaaagc tagtagtatt atgtttttaa ctaggcgttc
3181 gatcaggtgg atttcccctc cactagtttg gtcgatgagg ctaggaattc cccacgggtg
3241 accgtgtcct agcctgcgtg gcggccaacc cagcccactc actatttgtt ttcgcgccca
3301 gttgcaaaaa gtgtcggggc tgggacgcct ttttatagac atggtgtgaa gactcgcatg
3361 tgcttggttg tgattcctcc ggcccctgaa tgcggctaac cttaaccctg gagccttgtg
3421 tcacaaacca gtgatgataa ggtcgtaatg agcaattccg ggacgggacc gactactttg
3481 ggtgtccgtg tttcttattt ttcttattat tgtcttatgg tcacagcata tatataacat
3541 atactgtgat catgggatcc gactataagg accacgacgg agactacaag gatcatgata
3601 ttgattacaa ggacgacgat gacaagggag gaggaggaag cgtcttcaca ctcgaagatt
3661 tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag
3721 gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc
3781 tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga
3841 gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc
3901 atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca
3961 tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca
4021 ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg
4081 acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac
4141 gcattctggc gtagtaacaa ttggctggag cctcggtggc catgcttctt gccccttggg
4201 cctcccccca gcccctcctc cccttcctgc acccgtaccc ccgtggtctt tgaataaagt
4261 ctgaaccaca caaatggtcg ccgactcagt agatgttttc ttgggttaat tgaggcctga
4321 gtataaggtg acttatactt gtaatctatc taaacgggga acctctctag tagacaatcc
4381 cgtgctaaat tgtaggactg ccctttaata aatacttcta tatttaaaga ggtatttatg
4441 aaaagcggaa tttatcagat taaaaatact ttctctagag tcgacctgca gacgcgttca
4501 gatagatcgg tcggtgaaaa aaaaaaaaaa aaaaaaaaaa aagcggccgc ctagcataac
4561 cccttggggc ctctaaacgg gtcttgaggg gttttttggc tagcggccgg cctgtgcctt
4621 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
4681 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
4741 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
4801 atagcaggca tgctggggat gcggtgggct ctatggcggc gcgccacttt atgcttccgg
4861 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
4921 atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa gatacattga
4981 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
5041 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
5101 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
5161 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
5221 tgggcgtcgc ttggtcggtc atttcgaatc aggcaccggg cttgcgggtc atgcaccagg
5281 tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga
5341 aggggaggtt gcggggcgcg gaggtttcca ggaaggcggg caccccggcg cgctcggccg
5401 cctccactcc ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaca
5461 cgccgacggt ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt
5521 ccatctgttg ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct
5581 cggcgaacac cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc
5641 cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt
5701 gcagctcggt gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt
5761 agtcggcgaa cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg cgggtggcga
5821 ggcgcaccgt gggcttgtac tcggtcatgc gggagctttt tgcaaaagcc taggcctcca
5881 aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc ggcctctgca
5941 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
6001 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
6061 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
6121 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
6181 tgacacagag ctcgtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca
6241 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
6301 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
6361 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
6421 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
6481 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
6541 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg aggcgaaata
6601 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
6661 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
6721 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
6781 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
6841 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
6901 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
6961 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctggagcaa gacgtttccc
7021 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
7081 ttcatgatga tatattttta tct
//
