pCDNA6.2- GW- EmGFP- miR- Control

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  • Model: PVT10873
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pCDNA6.2-GW-EmGFP-miR-Control

Catalog No. PVT10873
Packing 2ug

 

pCDNA6.2-GW-EmGFP-miR-Control Information

Function Mammal Editing plasmids

Promoter: CMV promoter

Replicator: pUC Ori, F1 Ori, SV40 Ori

Terminator: HSV TK poly (A) signal

Plasmid classification: lactation cell plasmids; lactation editing plasmids; interference with RNA plasmid.

Plasmid size: 5759bp

Prokaryotic resistance: splendin Spe

Screening markers: oryzocin Blast

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic, LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 centigrade, aerobic, 5%CO2

Induction mode: no induction

5'sequencing primers: pEGFP-C-5

3'sequencing primers: HSVtk-pA-R:GTCTCCTTCCGTGTTTCAG

Note: can be connected to the miRNA fragment

 

 

pCDNA6.2-GW-EmGFP-miR-Control Description

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.

pCDNA6.2-GW/EmGFP-miR-Control.png

 

 

pCDNA6.2-GW-EmGFP-miR-Control Sequence

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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