LOCUS       sequence-443735-        6795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of TNRC6A-silencing domain (SD) with 
            S1332A/S1346A/S1616A/S1691A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6795)
  AUTHORS   Munakata F, Suzawa M, Ui-Tei K
  TITLE     Identification of Phosphorylated Amino Acids in Human TNRC6A 
            C-Terminal Region and Their Effects on the Interaction with the 
            CCR4-NOT Complex.
  JOURNAL   Genes (Basel). 2021 Feb 13;12(2):271. doi: 10.3390/genes12020271.
  PUBMED    33668648
REFERENCE   2  (bases 1 to 6795)
  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"; journalName: "Genes 
            (Basel)."; date: "2021-02-13"; volume: "12(2):271. doi"; pages: " 
            10.3390/genes12020271"
COMMENT     Sequence Label: pcDNA.5.1-FHS-TNRC6A-SD-S1332A/S1346A/S1616A/S...
FEATURES             Location/Qualifiers
     source          1..6795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(45..64)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        236..615
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        616..819
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     770..790
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    821..839
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    842..860
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     870..894
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      990..999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             999..1022
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1023..1049
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1053..1166
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     protein_bind    1727..1738
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     primer_bind     complement(2702..2721)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2703..2721)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2748..2765)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2754..2978
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3111..3130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3262..3309
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3317..4339
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3317..4339
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4469..4590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4506..4525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4560..4579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4639..4655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4639..4655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4652..4674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4663..4679
                     /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(4687..4693,4694..4711,4712..4717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4732..4753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4870..4887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5041..5629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5121..5140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5800..6591,6592..6660))
                     /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     6423..6442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6661..6765)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
       61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
      121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
      181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
      241 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      301 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      361 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      421 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      481 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      541 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      601 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      661 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      721 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      781 ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat
      841 ctccctatca gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg
      901 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      961 actctagcgt ttccaccgcg gtggcggccg ccaccatgga ttacaaggat gacgacgata
     1021 agtacccata cgatgttcca gattacgctt ctatggacga gaagaccacc ggctggcggg
     1081 gcggccacgt ggtggagggc ctggccggcg agctggagca gctgcgggcc aggctggagc
     1141 accaccctca gggccagcgg gagccctgcc cgggcggatc cccactccat cagccagcca
     1201 tgaagtcttt ccttgacaat gtcatgcccc acactacacc tgagctgcaa aaagggccat
     1261 caccaataaa tgctttcagc aacttcccta taggcttgaa ctcaaacttg aatgtaaata
     1321 tggatatgaa cagtattaaa gagccacagt caagactaag gaagtggacg acagtggaca
     1381 gcatttctgt gaacacatct ttggatcaaa actccagcaa acatggtgct atttcaagtg
     1441 gtttcaggct ggaagagtct ccatttgttc cctatgactt tatgaacagc agtacttcac
     1501 cagccgctcc tccaggttca ataggagatg gctggccacg tgccaaagct cctaacggct
     1561 ctagcagtgt taattggcca ccagaatttc gtcctggtga gccatggaaa ggttatccaa
     1621 acattgaccc tgaaactgac ccttacgtca ctcctggcag tgtcataaac aatctttcaa
     1681 ttaatactgt gcgggaagtt gaccacctca gggacaggaa cagtgggtca tcctcatcct
     1741 tgaacaccac gctgccttca actagtgcct ggtcatccat tcgtgcctcc aactacaacg
     1801 ttcccctcag cagtacagca caaagcactt cagccagaaa tagtgattcc aaattgacat
     1861 ggtctcctgg ttcagttaca aacacctctc tggctcatga gctgtggaag gtccctttgc
     1921 cacctaaaaa catcactgct ccgtcccgcc cacctccggg actgactggt cagaagccac
     1981 ccttgtctac gtgggataat tctccccttc gtataggtgg aggatgggga aattctgacg
     2041 ccagatatac cccaggttcc agctggggtg agagcagctc agggagaata acaaattggc
     2101 ttgttctaaa aaaccttaca cctcagatcg atggctcaac tctgcgcact ctgtgcatgc
     2161 agcacggccc gctgatcaca ttccacctga acctccctca cggaaatgct ctggtccgct
     2221 acagttcaaa agaagaggta gtgaaggcac aaaagtctct gcacatgtgt gtactgggga
     2281 atactactat tcttgctgag tttgccagtg aagaggagat cagtcgtttc tttgcacaaa
     2341 gccagtctct gacccctgct cccggctggc agtctctcgg gtccagccag agccggctgg
     2401 gctccctcga ctgttcccac tcattctcca gccggaccga tctcaatcac tggaatggtg
     2461 ctgggctgtc gggaactaac tgtggagacc ttcacggcac ttcactctgg gggaccccgc
     2521 attattccac aagcctgtgg ggtcccccaa gcagcagcga cccccgagga attagcagcc
     2581 cagctcccat taacgctttt ctttctgttg accacctggg tgggggtgga gagtccatgt
     2641 aactcgaggg ggggcccggt accttaatta attaaggtac caggtaagtg tacccaattc
     2701 gccctatagt gagtcgtatt acaattcact cgaaacccgc tgatcagcct cgactgtgcc
     2761 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2821 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2881 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2941 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3001 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3241 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     3301 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     3361 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3421 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     3481 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     3541 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     3601 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     3661 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     3721 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3781 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3841 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3901 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3961 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4021 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4081 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4141 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4201 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     4261 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4321 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     4381 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4441 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4501 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4561 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4621 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4681 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4741 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4801 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4861 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4921 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4981 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5041 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5101 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5161 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5221 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5281 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5341 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5401 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5461 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5521 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5581 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5641 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5701 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5761 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5821 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5881 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5941 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6001 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6061 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6121 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6181 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6241 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6301 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6361 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6421 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6481 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6541 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6601 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6661 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6721 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6781 agtgccacct gacgt
//
