Catalog No. PVT10873
Packing 2ug
Plasmid classification: lactation cell plasmids; lactation editing plasmids; interference with RNA plasmid.
pCDNA6.2-GW-EmGFP-miR-Control plasmid is connected to a small fragment based on the linearization vector of pcDNA6.2-GW/EmGFP-miR. If other miRNA fragments are needed, they can be linked by primer annealing and enzyme digestion. MiRNA was expressed immediately after transfection. The miRNA expressed in the nucleus was transferred to cytoplasm after being processed by endogenous cell mechanism (including Drosha enzyme), and further processed by Dicer enzyme. Then, the finished miRNA is integrated into RISC, where the function is similar to siRNA, so that the mRNA target is cut.
The BLOCK-iT Pol II miR RNAi expression vector kit with EmGFP has the advantages of the traditional RNAi vector, which is stable and capable of using the virus, as well as the functions of tissue specific expression and multiple target inhibition of the same transcriptional transcript. The pcDNA6.2-GW/EmGFP-miR vector containing the BLOCK-iT Pol II miR RNAi expression carrier kit with EmGFP is designed for the expression of artificial miRNA, which is engineered to have 100% homology with the sequence of the target gene and can cause the cutting of the target molecule. The carrier includes the lateral flanking sequence and ring sequence of endogenous miRNA, and endogenous miRNA can guide the removal of engineered miRNA from a longer Pol II transcript (pre-miRNA). Using the first class BLOCK-iT RNAi Designer, more than 70% of the recombinant body can produce more than 70% inhibition efficiency. The Pol II expression of Engineering miRNA can be realized as follows:
The CMV promoter was efficiently expressed under the immediate early promoter, and the tissue specific promoter and other regulated promoters could be selected through MultiSite Gateway recombination.
The expression of Emerald GFP (EmGFP) reporter gene is expressed by the same - CIS trans subunit in the pcDNA 6.2-GW/EmGFP-miR vector, which has a strong correlation between the expression of EmGFP and the inhibitory activity of the target miRNA.
Compatible with a variety of Invitrogen Gateway (DEST) carriers for gene expression, including lentivirus vectors for the stable transduction of split cells, non split cells and primary cell types, Flp-In TT systems for chromosome unit integration, and alternative reporter gene fusion constructs;
More than one engineered miRNA can be expressed in the same transcript, thus inhibiting multiple genes and producing synthetic phenotypes simultaneously.
LOCUS Exported 5759 bp ds-DNA circular SYN 19-SEP-2017
DEFINITION synthetic circular DNA
KEYWORDS pCDNA6.2-GW-EmGFP-miR-Control
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5759)
TITLE Direct Submission
JOURNAL Exported Sep 19, 2017
FEATURES Location/Qualifiers
source 1..5759
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 384..587
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 632..650
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 680..704
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 713..1432
/codon_start=1
/product="Emerald GFP"
/label=EmGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
ncRNA 1492..1518
/label=5' miR-155
/note="sequence upstream of the precursor of mouse miR-155
microRNA (Uva et al., 2013)"
/ncRNA_class="miRNA"
misc_feature 1519..1581
/label=Control
ncRNA 1582..1623
/label=3' miR-155
/note="sequence downstream of the precursor of mouse
miR-155 microRNA (Uva et al., 2013)"
/ncRNA_class="miRNA"
protein_bind complement(1652..1676)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
polyA_signal 1705..1976
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 2088..2516
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2530..2859
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2710..2845
/label=SV40 ori
/note="SV40 origin of replication"
promoter 2907..2954
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 2973..3371
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=Blast
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 3529..3659
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(3856..4444)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4540..5331)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=Spe
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
601 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagtcccaag
661 ctggctagtt aagctatcaa caagtttgta caaaaaagca ggctttaaaa ccatggtgag
721 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
781 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
841 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
901 caccttcacc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
961 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
1021 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
1081 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
1141 gtacaactac aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa
1201 ggtgaacttc aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
1261 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
1321 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
1381 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagctaagca
1441 cttcgtggcc gtcgatcgtt taaagggagg tagtgagtcg accagtggat cctggaggct
1501 tgctgaaggc tgtatgctgt gtcagtttgt caaatacccc gttttggcca ctgactgacg
1561 gggtattaca aactgacaca ggacacaagg cctgttacta gcactcacat ggaacaaatg
1621 gcccagatct ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggttgat
1681 ctagagggcc cgcggttcgc tgatggggga ggctaactga aacacggaag gagacaatac
1741 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
1801 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
1861 tgaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
1921 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcatcc
1981 cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc
2041 aaaatctctg atggatctgc gcagctgggg ctctaggggg tatccccacg cgccctgtag
2101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
2161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
2221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
2281 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
2341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
2401 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
2461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
2521 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
2581 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
2701 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
2761 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
2821 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
2881 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
2941 tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat
3001 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
3061 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
3121 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
3181 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
3241 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
3301 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
3361 gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
3421 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
3481 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
3541 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
3601 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
3661 ataccgtcgc tcttccgctg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
3721 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
3781 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
3841 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
3901 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
3961 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
4021 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
4081 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
4141 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
4201 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
4261 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
4321 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
4381 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
4441 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg acgcgtaact
4501 cacgttaagg gattttggtc atgggtggct cgacgagggt tatttgccga ctaccttggt
4561 gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg
4621 atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc
4681 cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac
4741 tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc
4801 gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg
4861 aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc
4921 ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag
4981 aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg
5041 aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc
5101 aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag
5161 ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac
5221 tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc
5281 aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca taatgtttaa
5341 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctccata acatcaaaca
5401 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtaccccaaa
5461 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
5521 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagct tacgaaccga
5581 acaggcttat gtccactggg ttcgtgcctt catccgtttc cacggtgtgc gtcacccggc
5641 aaccttgggt agcagcgaag tcgaggcatt tctgtcctgg ctggtctaga attgcatgaa
5701 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgc
//
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