LOCUS       sequence-180805-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of NHA-tagged human TNRC6C CED in mammalian 
            cells with mutated PAM2 motif.
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-658
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1089..1098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1095..1160
                     /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"
     CDS             1197..1223
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1203..1229
                     /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"
     polyA_signal    2295..2416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2306..2325)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2360..2379
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2602..3057
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2689..2708)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2899..2920
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3174..3531
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3174..3365
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3197..3217)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3382..3517
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3582..4376
                     /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(3636..3655)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4246..4265
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4440..4488
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4529..4548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4648..4670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4708..4726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4794..4898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4899..4967,4968..5759)
                     /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     complement(5117..5136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5930..6518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6419..6438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc caccatggac gcacaaacac gacgacgtga gcgtcgcgct gagaaacaag
     1141 ctcaatggaa agctgcaaac ccaccgctcg aggctgccca agcttacacc atggcctacc
     1201 catatgatgt tccagattac gcttcactcg acgcggatcc cgtcgaattc cgtgccaaat
     1261 ctgacagtga taaaatctca aatggctcta gcatcaactg gcccccagca gcccatccgg
     1321 gagttccatg gaaaggactg cagaatattg accctgagaa tgaccctgac gtcactcctg
     1381 gcagtgtccc cactgggcct accatcaaca ccaccatcca ggatgtcaac cgctacctcc
     1441 tcaagagtgg aggtaaactg tcagacatca aatcgacgtg gtcctctggc cctacctccc
     1501 acacgcaagc ctctctgtct catgaactat ggaaggtgcc cagaaacagt actgcaccca
     1561 cgaggccacc tccagggtta accaatccca agccctcctc cacctggggt gccagccccc
     1621 tcggctggac cagctcctac tcctcgggtt ctgcctggag caccgacacc tcaggaagaa
     1681 ccagcagctg gctcgttctt cgaaacctca ctccccagat tgatggttct acactgcgga
     1741 cattgtgttt gcaacatggg cctcttatca cattccacct gaatctgact caaggcaatg
     1801 ctgtggtccg gtacagctcc aaggaggagg ctgccaaggc ccagaagtct ctgcacatgt
     1861 gcgtcctggg aaacactacc atcctggccg agttcgctgg tgaagaagaa gtgaatcgct
     1921 tcttagccca aggccaggcg ctgccaccca cttccagctg gcagtccagc agcgcgtcca
     1981 gccagccgcg gctcagcgca gcgggcagct cccatggcct ggtacgcagc gacgctggcc
     2041 actggaacgc cccgtgcctg ggtggcaagg ggagcagtga gctgctgtgg ggcggggtgc
     2101 cccagtactc cagcagcctg tggggcccgc ccagcgccga cgacagcagg gtgataggca
     2161 gccccacgcc gctaaccacc ctgctgcctg gggacctgct cagcggggag tccctgtagg
     2221 cgctgccatc atcagcacca ggagagccgc ggccgcttcc ctttagtgag ggttaatgct
     2281 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     2341 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     2401 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     2461 gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg
     2521 atcgatccgg gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2581 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2641 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2701 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2761 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2821 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2881 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2941 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3001 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcct
     3061 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc
     3121 gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc
     3181 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3241 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3301 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3361 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3421 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3481 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat
     3541 tcttctgaca caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat
     3601 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3661 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3721 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3781 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3841 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3901 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3961 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4021 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4081 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4141 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4201 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4261 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4321 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4381 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca
     4441 ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga
     4501 taaggatccg cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4561 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4621 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4681 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4741 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4861 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4921 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4981 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5041 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5101 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5161 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5221 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5281 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5341 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5401 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5461 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5521 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5581 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5641 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5701 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5761 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5821 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5881 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5941 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6001 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6061 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6121 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6181 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6241 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6301 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6361 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6421 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6481 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6541 tacggttcct ggccttttgc tggccttttg ctcacatggc tcgacagatc t
//
