LOCUS       sequence-194409-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of GST-tagged human TNRC6C CED with 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-664
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1624..1641)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1648..2301)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1668..1690)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        complement(2353..3531)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2362..3300
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2385..2405)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3618..3814)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3626..3836)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3632..3767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3868..3884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3868..3884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3892..3908
                     /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(3916..3922,3923..3940,3941..3946))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3961..3982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4099..4116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4270..4858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4350..4369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5029..5820,5821..5889))
                     /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     5652..5671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5890..5994)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6062..6080
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6118..6140)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6240..6259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6276..6731)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6413..6434)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6625..6644
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6929..6951
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6943..6960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6944..6960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tttttttttt tttttttttt tttttttttt tttttttttt ggaggcaaac actttattat
       61 cctgaagttc agttacaatg tgacagtgga ggggacactg gtgagtgggg ggtgaagagg
      121 tggagggtga cagtggagct tcctttcaca cacaggcctg acatgattcc tcctgtctgc
      181 tccctcccac atccccagtc cccgtccagc aaggcaaatg tccaggcacc tgcctggccc
      241 taaaaagagt cacagtcctg accctcgtgg gggcagggca ggtgtcccga ggccacccag
      301 aaaaacagga gacacgtcgg tgatgttcgg gagtcaaacc atgtcccaaa agtcttaaga
      361 agaaaaacga gattccccag caaattcgtg ggaccccagg gccatgcctc ccagctctgg
      421 ccacctctgc ttgtcaaggg gcctcacagc caaggggatg caggggccgc tgtgcagtcg
      481 gctgaggcca gagtgagggg tgcaaggacc cagatgggga acactgctgt ttaaatatta
      541 aacagggatt tcttgtctcc cacgtgggag acctgatact ctcaagggtc ccaggtgggg
      601 accctcactc tagagtcgcg gccgcggctc tcctggtgct gatgatggca gcgcctacag
      661 ggactccccg ctgagcaggt ccccaggcag cagggtggtt agcggcgtgg ggctgcctat
      721 caccctgctg tcgtcggcgc tgggcgggcc ccacaggctg ctggagtact ggggcacccc
      781 gccccacagc agctcactgc tccccttgcc acccaggcac ggggcgttcc agtggccagc
      841 gtcgctgcgt accaggccat gggagctgcc cgctgcgctg agccgcggct ggctggacgc
      901 gctgctggac tgccagctgg aagtgggtgg cagcgcctgg ccttgggcta agaagcgatt
      961 cacttcttct tcaccagcga actcggccag gatggtagtg tttcccagga cgcacatgtg
     1021 cagagacttc tgggccttgg cagcctcctc cttggagctg taccggacca cagcattgcc
     1081 ttgagtcaga ttcaggtgga atgtgataag aggcccatgt tgcaaacaca atgtccgcag
     1141 tgtagaacca tcaatctggg gagtgaggtt tcgaagaacg agccagctgc tggttcttcc
     1201 tgaggtgtcg gtgctccagg cagaacccga ggagtaggag ctggtccagc cgagggggct
     1261 ggcaccccag gtggaggagg gcttgggatt ggttaaccct ggaggtggcc tcgtgggtgc
     1321 agtactgttt ctgggcacct tccatagttc atgagacaga gaggcttgcg tgtgggaggt
     1381 agggccagag gaccacgtcg atttgatgtc tgacagttta cctccactct tgaggaggta
     1441 gcggttgaca tcctggatgg tggtgttgat ggtaggccca gtggggacac tgccaggagt
     1501 gacgtcaggg tcattctcag ggtcaatatt ctgcagtcct ttccatggaa ctcccggatg
     1561 ggctgctggg ggccagttga tgctagagcc atttgagatt ttatcactgt cagatttggc
     1621 acgggatcca cgcggaacca gatccgattt tggaggatgg tcgccaccac caaacgtggc
     1681 ttgccagccc tgcaaaggcc atgctatata cttgctggat ttcaagtact tatcaatttg
     1741 tgggatagct tcaatacgtt ttttaaaaca aactaatttt gggaacgcat ccaggcacat
     1801 tgggtccatg tataaaacaa catcaagagc gtcatacaac atgaagtcag gatgggttac
     1861 atgatcacca tttaaatatg ttttatgaca taaacgatct tcgaacattt tcagcatttc
     1921 aggtagcttg ctaagaaaat caactttgag agtttcaaag tctttactat atgcaattct
     1981 cgaaacaccg tatctaatat ccaaaaccgc tccttcaagc attgaaatct ctgcacgctc
     2041 ttttggacaa ccacccaaca tgttgtgctt gtcagctata taacgtatga tggccataga
     2101 ctgtgttaat ttaacatcac catcaatata ataaggaaga ttgggaaact ccaaacccaa
     2161 ttcaaacttt ttgtttcgcc atttatcacc ttcatcgcgc tcatacaaat gctcttcata
     2221 tttttcttca agatattcca aaagaagtcg agtgggttgc acaaggccct taattttcca
     2281 ataacctagt ataggggcca ttgtagatcc agcacaatgg atctcgaggt cgagggatct
     2341 ctagagaatt cctcacgaca cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg
     2401 cttgagaatg aaccaagatc caaactcaaa aagggcaaat tccaaggaga attacatcaa
     2461 gtgccaagct ggcctaactt cagtctccac ccactcagtg tggggaaact ccatcgcata
     2521 aaacccctcc ccccaaccta aagacgacgt actccaaaag ctcgagaact aatcgaggtg
     2581 cctggacggc gcccggtact ccgtggagtc acatgaagcg acggctgagg acggaaaggc
     2641 ccttttcctt tgtgtgggtg actcacccgc ccgctctccc gagcgccgcg tcctccattt
     2701 tgagctccct gcagcagggc cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga
     2761 ccgggccagc cttgccgccc agggcggggc gatacacggc ggcgcgaggc caggcaccag
     2821 agcaggccgg ccagcttgag actacccccg tccgattctc ggtggccgcg ctcgcaggcc
     2881 ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg tcgccgcccg cggccccaaa
     2941 aaccgaaata ccagtgtgca gatcttggcc cgcatttaca agactatctt gccagaaaaa
     3001 aagcgtcgca gcaggtcatc aaaaatttta aatggctaga gacttatcga aagcagcgag
     3061 acaggcgcga aggtgccacc agattcgcac gcggcggccc cagcgcccag gccaggcctc
     3121 aactcaagca cgaggcgaag gggctcctta agcgcaaggc ctcgaactct cccacccact
     3181 tccaacccga agctcgggat caagaatcac gtactgcagc caggtggaag taattcaagg
     3241 cacgcaaggg ccataacccg taaagaggcc aggcccgcgg gaaccacaca cggcacttac
     3301 ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat
     3361 atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca
     3421 gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt
     3481 ctcggggact gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc caattcccac
     3541 tcctttcaag acctagaagg tccattagct gcaaagattc ctctctgttt aaaactttat
     3601 ccatctttgc aaagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     3661 tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     3721 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     3781 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     3841 gagggaatta agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3901 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3961 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4081 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4141 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4201 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4261 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4321 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4381 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4441 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4501 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4561 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4621 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4681 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4741 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4801 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4861 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4921 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4981 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5041 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5101 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5161 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5221 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5281 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5341 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5401 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5461 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5521 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5581 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5641 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5701 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5761 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5821 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5881 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5941 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6001 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     6061 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     6121 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     6181 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     6241 ggcatcagag cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt
     6301 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg
     6361 caaaatccct tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg
     6421 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6481 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg
     6541 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6601 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct
     6661 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6721 acagggcgcg tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta
     6781 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6841 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     6901 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     6961 gaattc
//
