LOCUS       sequence-194028-        6591 bp    DNA     circular SYN 15-APR-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 Apr 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        65..256
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(373..828)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(510..531)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     722..741
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1014..1135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1105..1124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2201..2227)
                     /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(2207..2233)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2270..2335)
                     /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      2332..2341
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2345..2363)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2406..2425)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2408..2540
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(2680..2704)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2701..2912)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2730..2750)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2913..3292
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3503..4091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3583..3602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4262..5053,5054..5122))
                     /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     4885..4904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5123..5227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5295..5313
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5351..5373)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5473..5492
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    5533..5581
                     /note="synthetic polyadenylation signal"
     CDS             complement(5645..6439)
                     /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(5756..5775)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6366..6385
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6490..6591,1..256))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6504..6591,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg
      241 gttctttccg cctcagaagg tacctaacca agttcctctt tcagaggtta tttcaggcca
      301 tggtgctgcg cagatccgcg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
      361 taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
      421 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
      481 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
      541 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
      601 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
      661 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
      721 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
      781 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt
      841 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc
      901 cggatcgatc cttatcggat tttaccacat ttgtagaggt tttacttgct ttaaaaaacc
      961 tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     1021 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1081 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     1141 tctgctcgaa gcattaaccc tcactaaagg gaagcggccg cggctctcct ggtgctgatg
     1201 atggcagcgc ctacagggac tccccgctga gcaggtcccc aggcagcagg gtggttagcg
     1261 gcgtggggct gcctatcacc ctgctgtcgt cggcgctggg cgggccccac aggctgctgg
     1321 agtactgggg caccccgccc cacagcagct cactgctccc cttgccaccc aggcacgggg
     1381 cgttccagtg gccagcgtcg ctgcgtacca ggccatggga gctgcccgct gcgctgagcc
     1441 gcggctggct ggacgcgctg ctggactgcc agctggaagt gggtggcagc gcctggcctt
     1501 gggctaagaa gcgattcact tcttcttcac cagcgaactc ggccaggatg gtagtgtttc
     1561 ccaggacgca catgtgcaga gacttctggg ccttggcagc ctcctccttg gagctgtacc
     1621 ggaccacagc attgccttga gtcagattca ggtggaatgt gataagaggc ccatgttgca
     1681 aacacaatgt ccgcagtgta gaaccatcaa tctggggagt gaggtttcga agaacgagcc
     1741 agctgctggt tcttcctgag gtgtcggtgc tccaggcaga acccgaggag taggagctgg
     1801 tccagccgag ggggctggca ccccaggtgg aggagggctt gggattggtt aaccctggag
     1861 gtggcctcgt gggtgcagta ctgtttctgg gcaccttcca tagttcatga gacagagagg
     1921 cttgcgtgtg ggaggtaggg ccagaggacc acgtcgattt gatgtctgac agtttacctc
     1981 cactcttgag gaggtagcgg ttgacatcct ggatggtggt gttgatggta ggcccagtgg
     2041 ggacactgcc aggagtgacg tcagggtcat tctcagggtc aatattctgc agtcctttcc
     2101 atggaactcc cggatgggct gctgggggcc agttgatgct agagccattt gagattttat
     2161 cactgtcaga tttggcacgg aattcgacgg gatccgcgtc gagtgaagcg taatctggaa
     2221 catcatatgg gtaggccatg gtgtaagctt gggcagcctc gagcggtggg tttgcagctt
     2281 tccattgagc ttgtttctca gcgcgacgct cacgtcgtcg tgtttgtgcg tccatggtgg
     2341 ctagcctata gtgagtcgta ttaagtactc tagccttaag agctgtaatt gaactgggag
     2401 tggacacctg tggagagaaa ggcaaagtgg atgtcagtaa gaccaatagg tgcctatcag
     2461 aaacgcaaga gtcttctctg tctcgacaag cccagtttct attggtctcc ttaaacctgt
     2521 cttgtaacct tgatacttac ctgcccagtg cctcacgacc aacttctgca aggctacctt
     2581 aagagagtca ctgcagctta agttcgagac tgttgtgtca gaagcactga ctgcgttagc
     2641 aatttaactg tgataaacta ccgcaataaa gcttctagtg atctgacggt tcactaaacg
     2701 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaacggggc
     2761 gggcgatcgc agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     2821 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     2881 cgcccattgg tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     2941 atgtactgcc aagtaggaaa gtcccgtaag gtcatgtact gggcataatg ccaggcgggc
     3001 catttaccgt cattgacgtc aatagggggc ggacttggca tatgatacac ttgatgtact
     3061 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     3121 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     3181 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     3241 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg ccaacatggc
     3301 ggtcatattg gacatgagcc aatataaatg tacatattat gatatagata caacgtatgc
     3361 aatggccaat agccaatatt gatttatgct atataaccaa tgaataatat ggctaatggc
     3421 caatattgaa gatctgtcga gccatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3481 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3541 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3601 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3661 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3721 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3781 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3841 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3901 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3961 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4021 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4081 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4141 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4201 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4261 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4321 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4381 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4441 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4501 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4561 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4621 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4681 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4741 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4801 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4861 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4921 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4981 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5041 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5101 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5161 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5221 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5281 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     5341 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     5401 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     5461 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatacg cggatcctta
     5521 tcgctatcga ttcacacaaa aaaccaacac acagatgtaa tgaaaataaa gatattttat
     5581 tgcggccatc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc
     5641 ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
     5701 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
     5761 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga
     5821 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg
     5881 tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg
     5941 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
     6001 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
     6061 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
     6121 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
     6181 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
     6241 gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa
     6301 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
     6361 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
     6421 atccatcttg ttcaatcatg gtggctctag ccttaagttc gagactgttg tgtcagaaga
     6481 atcaagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag
     6541 ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc a
//
