LOCUS       sequence-194509-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of NHA-tagged human TNRC6C 7W7A CED with 
            seven mutated W-motifs and mutated PAM2 motif in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  AUTHORS   Chekulaeva M, Mathys H, Zipprich JT, Attig J, Colic M, Parker R, 
            Filipowicz W
  TITLE     miRNA repression involves GW182-mediated recruitment of CCR4-NOT 
            through conserved W-containing motifs.
  JOURNAL   Nat Struct Mol Biol. 2011 Oct 7;18(11):1218-26. doi: 
            10.1038/nsmb.2166.
  PUBMED    21984184
REFERENCE   2  (bases 1 to 6591)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nsmb"; 
            journalName: "Nat Struct Mol Biol"; date: "2011-10-7- 7"; volume: 
            "18"; issue: "11"; pages: "1218-26"
COMMENT     Sequence Label: pMCh-660
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..216
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     173..193
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(333..788)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(470..491)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     682..701
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    974..1095
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1011..1030)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1065..1084
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2161..2187)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             complement(2167..2193)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2230..2295)
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     regulatory      2292..2301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2305..2323)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2366..2385)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2368..2500
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(2640..2664)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2661..2872)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2690..2710)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2873..3252
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3463..4051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3543..3562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4222..5013,5014..5082))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4845..4864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5083..5187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5255..5273
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5311..5333)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5433..5452
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    5493..5541
                     /note="synthetic polyadenylation signal"
     CDS             complement(5605..6399)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5716..5735)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6326..6345
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6450..6591,1..216))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6464..6591,1..8)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
       61 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
      121 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
      181 tgacacacat tccacagctg gttctttccg cctcagaagg tacctaacca agttcctctt
      241 tcagaggtta tttcaggcca tggtgctgcg cagatccgcg tatgcggtgt gaaataccgc
      301 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa
      361 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
      421 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
      481 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
      541 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
      601 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
      661 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
      721 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
      781 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      841 tcttcgctat tacgccagcc cggatcgatc cttatcggat tttaccacat ttgtagaggt
      901 tttacttgct ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc
      961 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1021 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1081 catcaatgta tcttatcatg tctgctcgaa gcattaaccc tcactaaagg gaagcggccg
     1141 cggctctcct ggtgctgatg atggcagcgc ctacagggac tccccgctga gcaggtcccc
     1201 aggcagcagg gtggttagcg gcgtggggct gcctatcacc ctgctgtcgt cggcgctggg
     1261 cgggccggcc aggctgctgg agtactgggg caccccgccg gccagcagct cactgctccc
     1321 cttgccaccc aggcacgggg cgttccagtg gccagcgtcg ctgcgtacca ggccatggga
     1381 gctgcccgct gcgctgagcc gcggctggct ggacgcgctg ctggactggg cgctggaagt
     1441 gggtggcagc gcctggcctt gggctaagaa gcgattcact tcttcttcac cagcgaactc
     1501 ggccaggatg gtagtgtttc ccaggacgca catgtgcaga gacttctggg ccttggcagc
     1561 ctcctccttg gagctgtacc ggaccacagc attgccttga gtcagattca ggtggaatgt
     1621 gataagaggc ccatgttgca aacacaatgt ccgcagtgta gaaccatcaa tctggggagt
     1681 gaggtttcga agaacgagcc agctgctggt tcttcctgag gtgtcggtgc tggcggcaga
     1741 acccgaggag taggagctgg tggcgccgag ggggctggca ccggcggtgg aggagggctt
     1801 gggattggtt aaccctggag gtggcctcgt gggtgcagta ctgtttctgg gcaccttcca
     1861 tagttcatga gacagagagg cttgcgtgtg ggaggtaggg ccagaggagg ccgtcgattt
     1921 gatgtctgac agtttacctc cactcttgag gaggtagcgg ttgacatcct ggatggtggt
     1981 gttgatggta ggcccagtgg ggacactgcc aggagtgacg tcagggtcat tctcagggtc
     2041 aatattctgc agtcctttcc atggaactcc cggatgggct gctgggggcc agttgatgct
     2101 agagccattt gagattttat cactgtcaga tttggcacgg aattcgacgg gatccgcgtc
     2161 gagtgaagcg taatctggaa catcatatgg gtaggccatg gtgtaagctt gggcagcctc
     2221 gagcggtggg tttgcagctt tccattgagc ttgtttctca gcgcgacgct cacgtcgtcg
     2281 tgtttgtgcg tccatggtgg ctagcctata gtgagtcgta ttaagtactc tagccttaag
     2341 agctgtaatt gaactgggag tggacacctg tggagagaaa ggcaaagtgg atgtcagtaa
     2401 gaccaatagg tgcctatcag aaacgcaaga gtcttctctg tctcgacaag cccagtttct
     2461 attggtctcc ttaaacctgt cttgtaacct tgatacttac ctgcccagtg cctcacgacc
     2521 aacttctgca aggctacctt aagagagtca ctgcagctta agttcgagac tgttgtgtca
     2581 gaagcactga ctgcgttagc aatttaactg tgataaacta ccgcaataaa gcttctagtg
     2641 atctgacggt tcactaaacg agctctgctt atatagacct cccaccgtac acgcctaccg
     2701 cccatttgcg tcaacggggc gggcgatcgc agttgttacg acattttgga aagtcccgtt
     2761 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     2821 gtgagtcaaa ccgctatcca cgcccattgg tgtactgcca aaaccgcatc accatggtaa
     2881 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccgtaag gtcatgtact
     2941 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc ggacttggca
     3001 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     3061 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     3121 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     3181 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     3241 ataatcaatg ccaacatggc ggtcatattg gacatgagcc aatataaatg tacatattat
     3301 gatatagata caacgtatgc aatggccaat agccaatatt gatttatgct atataaccaa
     3361 tgaataatat ggctaatggc caatattgaa gatctgtcga gccatgtgag caaaaggcca
     3421 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3481 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3541 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3601 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3661 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3721 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3781 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3841 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3901 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3961 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4021 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4081 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4141 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4201 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4261 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4321 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4381 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4441 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4501 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4561 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4621 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4681 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4741 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4801 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4861 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4921 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4981 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5041 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5101 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5161 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     5221 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     5281 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5341 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5401 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     5461 gcaccatacg cggatcctta tcgctatcga ttcacacaaa aaaccaacac acagatgtaa
     5521 tgaaaataaa gatattttat tgcggccatc gtgatggcag gttgggcgtc gcttggtcgg
     5581 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     5641 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     5701 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     5761 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     5821 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     5881 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     5941 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     6001 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     6061 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     6121 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     6181 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     6241 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     6301 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     6361 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg gtggctctag ccttaagttc
     6421 gagactgttg tgtcagaaga atcaagcttt ttgcaaaagc ctaggcctcc aaaaaagcct
     6481 cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa
     6541 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag g
//
