LOCUS       sequence-194411-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of GST-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 6966)
  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 6966)
  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-665
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1627..1644)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1651..2304)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1671..1693)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        complement(2356..3534)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2365..3303
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2388..2408)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3621..3817)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3629..3839)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3635..3770
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3871..3887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3871..3887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3884..3906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3895..3911
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3919..3925,3926..3943,3944..3949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3964..3985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4102..4119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4273..4861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4353..4372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5032..5823,5824..5892))
                     /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     5655..5674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5893..5997)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6065..6083
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6121..6143)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6243..6262
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6279..6734)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6416..6437)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6628..6647
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6932..6954
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6946..6963
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6947..6963
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttcttttttt tttttttttt tttttttttt tttttttttt tttggaggca aacactttat
       61 tatcctgaag ttcagttaca atgtgacagt ggaggggaca ctggtgagtg gggggtgaag
      121 aggtggaggg tgacagtgga gcttcctttc acacacaggc ctgacatgat tcctcctgtc
      181 tgctccctcc cacatcccca gtccccgtcc agcaaggcaa atgtccaggc acctgcctgg
      241 ccctaaaaag agtcacagtc ctgaccctcg tgggggcagg gcaggtgtcc cgaggccacc
      301 cagaaaaaca ggagacacgt cggtgatgtt cgggagtcaa accatgtccc aaaagtctta
      361 agaagaaaaa cgagattccc cagcaaattc gtgggacccc agggccatgc ctcccagctc
      421 tggccacctc tgcttgtcca ggggcctcac agccaagggg atgcaggggc cgctgtgcag
      481 tcggctgagg ccagagtgag gggtgcaagg acccagatgg ggaacactgc tgtttaaata
      541 ttaaacaggg atttcttgtc tcccacgtgg gagacctgat actctcaagg gtcccaggtg
      601 gggaccctca ctctagagtc gcggccgcgg ctctcctggt gctgatgatg gcagcgccta
      661 cagggactcc ccgctgagca ggtccccagg cagcagggtg gttagcggcg tggggctgcc
      721 tatcaccctg ctgtcgtcgg cgctgggcgg gccggccagg ctgctggagt actggggcac
      781 cccgccggcc agcagctcac tgctcccctt gccacccagg cacggggcgt tccagtggcc
      841 agcgtcgctg cgtaccaggc catgggagct gcccgctgcg ctgagccgcg gctggctgga
      901 cgcgctgctg gactgggcgc tggaagtggg tggcagcgcc tggccttggg ctaagaagcg
      961 attcacttct tcttcaccag cgaactcggc caggatggta gtgtttccca ggacgcacat
     1021 gtgcagagac ttctgggcct tggcagcctc ctccttggag ctgtaccgga ccacagcatt
     1081 gccttgagtc agattcaggt ggaatgtgat aagaggccca tgttgcaaac acaatgtccg
     1141 cagtgtagaa ccatcaatct ggggagtgag gtttcgaaga acgagccagc tgctggttct
     1201 tcctgaggtg tcggtgctgg cggcagaacc cgaggagtag gagctggtgg cgccgagggg
     1261 gctggcaccg gcggtggagg agggcttggg attggttaac cctggaggtg gcctcgtggg
     1321 tgcagtactg tttctgggca ccttccatag ttcatgagac agagaggctt gcgtgtggga
     1381 ggtagggcca gaggaggccg tcgatttgat gtctgacagt ttacctccac tcttgaggag
     1441 gtagcggttg acatcctgga tggtggtgtt gatggtaggc ccagtgggga cactgccagg
     1501 agtgacgtca gggtcattct cagggtcaat attctgcagt cctttccatg gaactcccgg
     1561 atgggctgct gggggccagt tgatgctaga gccatttgag attttatcac tgtcagattt
     1621 ggcacgggat ccacgcggaa ccagatccga ttttggagga tggtcgccac caccaaacgt
     1681 ggcttgccag ccctgcaaag gccatgctat atacttgctg gatttcaagt acttatcaat
     1741 ttgtgggata gcttcaatac gttttttaaa acaaactaat tttgggaacg catccaggca
     1801 cattgggtcc atgtataaaa caacatcaag agcgtcatac aacatgaagt caggatgggt
     1861 tacatgatca ccatttaaat atgttttatg acataaacga tcttcgaaca ttttcagcat
     1921 ttcaggtagc ttgctaagaa aatcaacttt gagagtttca aagtctttac tatatgcaat
     1981 tctcgaaaca ccgtatctaa tatccaaaac cgctccttca agcattgaaa tctctgcacg
     2041 ctcttttgga caaccaccca acatgttgtg cttgtcagct atataacgta tgatggccat
     2101 agactgtgtt aatttaacat caccatcaat ataataagga agattgggaa actccaaacc
     2161 caattcaaac tttttgtttc gccatttatc accttcatcg cgctcataca aatgctcttc
     2221 atatttttct tcaagatatt ccaaaagaag tcgagtgggt tgcacaaggc ccttaatttt
     2281 ccaataacct agtatagggg ccattgtaga tccagcacaa tggatctcga ggtcgaggga
     2341 tctctagaga attcctcacg acacctgaaa tggaagaaaa aaactttgaa ccactgtctg
     2401 aggcttgaga atgaaccaag atccaaactc aaaaagggca aattccaagg agaattacat
     2461 caagtgccaa gctggcctaa cttcagtctc cacccactca gtgtggggaa actccatcgc
     2521 ataaaacccc tccccccaac ctaaagacga cgtactccaa aagctcgaga actaatcgag
     2581 gtgcctggac ggcgcccggt actccgtgga gtcacatgaa gcgacggctg aggacggaaa
     2641 ggcccttttc ctttgtgtgg gtgactcacc cgcccgctct cccgagcgcc gcgtcctcca
     2701 ttttgagctc cctgcagcag ggccgggaag cggccatctt tccgctcacg caactggtgc
     2761 cgaccgggcc agccttgccg cccagggcgg ggcgatacac ggcggcgcga ggccaggcac
     2821 cagagcaggc cggccagctt gagactaccc ccgtccgatt ctcggtggcc gcgctcgcag
     2881 gccccgcctc gccgaacatg tgcgctggga cgcacgggcc ccgtcgccgc ccgcggcccc
     2941 aaaaaccgaa ataccagtgt gcagatcttg gcccgcattt acaagactat cttgccagaa
     3001 aaaaagcgtc gcagcaggtc atcaaaaatt ttaaatggct agagacttat cgaaagcagc
     3061 gagacaggcg cgaaggtgcc accagattcg cacgcggcgg ccccagcgcc caggccaggc
     3121 ctcaactcaa gcacgaggcg aaggggctcc ttaagcgcaa ggcctcgaac tctcccaccc
     3181 acttccaacc cgaagctcgg gatcaagaat cacgtactgc agccaggtgg aagtaattca
     3241 aggcacgcaa gggccataac ccgtaaagag gccaggcccg cgggaaccac acacggcact
     3301 tacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     3361 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     3421 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     3481 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agccaattcc
     3541 cactcctttc aagacctaga aggtccatta gctgcaaaga ttcctctctg tttaaaactt
     3601 tatccatctt tgcaaagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac
     3661 ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt
     3721 cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt
     3781 aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     3841 ggggagggaa ttaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3901 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3961 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4081 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4681 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6001 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     6061 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6121 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6181 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6241 tgcggcatca gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt
     6301 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat
     6361 cggcaaaatc ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt
     6421 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6481 ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag
     6541 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6601 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc
     6661 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6721 gctacagggc gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc
     6781 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     6841 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6901 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     6961 agtgaa
//
