LOCUS       sequence-194475-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of NHA-tagged human TNRC6C CED 7W7A with 
            seven mutated W-motifs 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-607
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..203
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     160..180
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(320..775)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(457..478)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     669..688
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    961..1082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(998..1017)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1052..1071
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2148..2174)
                     /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(2154..2180)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2217..2282)
                     /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      2279..2288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2292..2310)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2353..2372)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2355..2487
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(2627..2651)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2648..2859)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2677..2697)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2860..3239
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3450..4038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3530..3549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4209..5000,5001..5069))
                     /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     4832..4851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5070..5174)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5242..5260
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5298..5320)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5420..5439
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    5480..5528
                     /note="synthetic polyadenylation signal"
     CDS             complement(5592..6386)
                     /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(5703..5722)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6313..6332
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6437..6591,1..203))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6451..6586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc
       61 ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt
      121 gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc
      181 acagctggtt ctttccgcct cagaaggtac ctaaccaagt tcctctttca gaggttattt
      241 caggccatgg tgctgcgcag atccgcgtat gcggtgtgaa ataccgcaca gatgcgtaag
      301 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
      361 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
      421 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
      481 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
      541 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
      601 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
      661 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
      721 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt
      781 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      841 gccagcccgg atcgatcctt atcggatttt accacatttg tagaggtttt acttgcttta
      901 aaaaacctcc cacatctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
      961 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1021 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1081 tatcatgtct gctcgaagca ttaaccctca ctaaagggaa gcggccgcgg ctctcctggt
     1141 gctgatgatg gcagcgccta cagggactcc ccgctgagca ggtccccagg cagcagggtg
     1201 gttagcggcg tggggctgcc tatcaccctg ctgtcgtcgg cgctgggcgg gccggccagg
     1261 ctgctggagt actggggcac cccgccggcc agcagctcac tgctcccctt gccacccagg
     1321 cacggggcgt tccagtggcc agcgtcgctg cgtaccaggc catgggagct gcccgctgcg
     1381 ctgagccgcg gctggctgga cgcgctgctg gactgggcgc tggaagtggg tggcagcgcc
     1441 tggccttggg ctaagaagcg attcacttct tcttcaccag cgaactcggc caggatggta
     1501 gtgtttccca ggacgcacat gtgcagagac ttctgggcct tggcagcctc ctccttggag
     1561 ctgtaccgga ccacagcatt gccttgagtc agattcaggt ggaatgtgat aagaggccca
     1621 tgttgcaaac acaatgtccg cagtgtagaa ccatcaatct ggggagtgag gtttcgaaga
     1681 acgagccagc tgctggttct tcctgaggtg tcggtgctgg cggcagaacc cgaggagtag
     1741 gagctggtgg cgccgagggg gctggcaccg gcggtggagg agggcttggg attggttaac
     1801 cctggaggtg gcctcgtggg tgcagtactg tttctgggca ccttccatag ttcatgagac
     1861 agagaggctt gcgtgtggga ggtagggcca gaggaggccg tcgatttgat gtctgacagt
     1921 ttacctccac tcttgaggag gtagcggttg acatcctgga tggtggtgtt gatggtaggc
     1981 ccagtgggga cactgccagg agtgacgtca gggtcattct cagggtcaat attctgcagt
     2041 cctttccatg gaactcccgg atggaattct gggggccagt tgatgctaga gccatttgag
     2101 attttatcac tgtcagattt ggcacggaat tcgacgggat ccgcgtcgag tgaagcgtaa
     2161 tctggaacat catatgggta ggccatggtg taagcttggg cagcctcgag cggtgggttt
     2221 gcagctttcc attgagcttg tttctcagcg cgacgctcac gtcgtcgtgt ttgtgcgtcc
     2281 atggtggcta gcctatagtg agtcgtatta agtactctag ccttaagagc tgtaattgaa
     2341 ctgggagtgg acacctgtgg agagaaaggc aaagtggatg tcagtaagac caataggtgc
     2401 ctatcagaaa cgcaagagtc ttctctgtct cgacaagccc agtttctatt ggtctcctta
     2461 aacctgtctt gtaaccttga tacttacctg cccagtgcct cacgaccaac ttctgcaagg
     2521 ctaccttaag agagtcactg cagcttaagt tcgagactgt tgtgtcagaa gcactgactg
     2581 cgttagcaat ttaactgtga taaactaccg caataaagct tctagtgatc tgacggttca
     2641 ctaaacgagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     2701 acggggcggg cgatcgcagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     2761 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     2821 ctatccacgc ccattggtgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     2881 tacgtagatg tactgccaag taggaaagtc ccgtaaggtc atgtactggg cataatgcca
     2941 ggcgggccat ttaccgtcat tgacgtcaat agggggcgga cttggcatat gatacacttg
     3001 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     3061 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     3121 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     3181 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgcca
     3241 acatggcggt catattggac atgagccaat ataaatgtac atattatgat atagatacaa
     3301 cgtatgcaat ggccaatagc caatattgat ttatgctata taaccaatga ataatatggc
     3361 taatggccaa tattgaagat ctgtcgagcc atgtgagcaa aaggccagca aaaggccagg
     3421 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3481 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3541 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3601 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3661 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3721 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3781 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3841 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3901 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3961 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4021 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4081 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4141 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4201 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4261 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4321 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4381 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4441 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4501 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4561 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4621 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4681 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4741 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4801 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4861 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4921 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4981 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5041 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5101 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5161 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5221 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     5281 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     5341 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     5401 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatacgcgg
     5461 atccttatcg ctatcgattc acacaaaaaa ccaacacaca gatgtaatga aaataaagat
     5521 attttattgc ggccatcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc
     5581 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     5641 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     5701 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     5761 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     5821 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
     5881 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     5941 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     6001 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     6061 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     6121 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     6181 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
     6241 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     6301 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     6361 gcgtgcaatc catcttgttc aatcatggtg gctctagcct taagttcgag actgttgtgt
     6421 cagaagaatc aagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     6481 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     6541 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc g
//
