LOCUS       sequence-443601-        7581 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 7581)
  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 7581)
  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: 
            pcDNA3.1-myc-GFP-TNRC6A-SD-S1332A/S1346A/S1616A/S1691A
FEATURES             Location/Qualifiers
     source          1..7581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             909..938
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(948..950,951..953,954..1664)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(948..950,951..953,954..1664)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(993..1014)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1254..1273)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1601..1622
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2225..2236
                     /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(3172..3189)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3178..3402
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3448..3876
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3535..3554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3745..3766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3885..3905)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3890..4219
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4070..4205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4132..4151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4286..5080
                     /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(4340..4359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4950..4969
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5254..5375
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5291..5310)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5345..5364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5424..5440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5424..5440)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5437..5459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5448..5464
                     /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(5472..5478,5479..5496,5497..5502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5517..5538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5655..5672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5826..6414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5906..5925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6585..7376,7377..7445))
                     /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     7208..7227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7446..7550)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 ccaccatgga acaaaaactc atctcagaag aggatctgtc cggcggcatg gtgagcaagg
      961 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1021 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1081 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1141 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1201 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1261 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1321 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1381 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1441 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1501 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1561 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1621 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccggc ggcaagcttc
     1681 cactccatca gccagccatg aagtctttcc ttgacaatgt catgccccac actacacctg
     1741 agctgcaaaa agggccatca ccaataaatg ctttcagcaa cttccctata ggcttgaact
     1801 caaacttgaa tgtaaatatg gatatgaaca gtattaaaga gccacagtca agactaagga
     1861 agtggacgac agtggacagc atttctgtga acacatcttt ggatcaaaac tccagcaaac
     1921 atggtgctat ttcaagtggt ttcaggctgg aagagtctcc atttgttccc tatgacttta
     1981 tgaacagcag tacttcacca gccgctcctc caggttcaat aggagatggc tggccacgtg
     2041 ccaaagctcc taacggctct agcagtgtta attggccacc agaatttcgt cctggtgagc
     2101 catggaaagg ttatccaaac attgaccctg aaactgaccc ttacgtcact cctggcagtg
     2161 tcataaacaa tctttcaatt aatactgtgc gggaagttga ccacctcagg gacaggaaca
     2221 gtgggtcatc ctcatccttg aacaccacgc tgccttcaac tagtgcctgg tcatccattc
     2281 gtgcctccaa ctacaacgtt cccctcagca gtacagcaca aagcacttca gccagaaata
     2341 gtgattccaa attgacatgg tctcctggtt cagttacaaa cacctctctg gctcatgagc
     2401 tgtggaaggt ccctttgcca cctaaaaaca tcactgctcc gtcccgccca cctccgggac
     2461 tgactggtca gaagccaccc ttgtctacgt gggataattc tccccttcgt ataggtggag
     2521 gatggggaaa ttctgacgcc agatataccc caggttccag ctggggtgag agcagctcag
     2581 ggagaataac aaattggctt gttctaaaaa accttacacc tcagatcgat ggctcaactc
     2641 tgcgcactct gtgcatgcag cacggcccgc tgatcacatt ccacctgaac ctccctcacg
     2701 gaaatgctct ggtccgctac agttcaaaag aagaggtagt gaaggcacaa aagtctctgc
     2761 acatgtgtgt actggggaat actactattc ttgctgagtt tgccagtgaa gaggagatca
     2821 gtcgtttctt tgcacaaagc cagtctctga cccctgctcc cggctggcag tctctcgggt
     2881 ccagccagag ccggctgggc tccctcgact gttcccactc attctccagc cggaccgatc
     2941 tcaatcactg gaatggtgct gggctgtcgg gaactaactg tggagacctt cacggcactt
     3001 cactctgggg gaccccgcat tattccacaa gcctgtgggg tcccccaagc agcagcgacc
     3061 cccgaggaat tagcagccca gctcccatta acgcttttct ttctgttgac cacctgggtg
     3121 ggggtggaga gtccatgtaa tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     3181 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3241 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3301 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3361 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3421 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3481 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3541 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3601 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3661 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3721 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3841 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3901 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3961 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4021 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4141 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4201 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4261 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4321 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4381 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4441 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4501 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4561 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4621 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4681 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4741 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4801 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4861 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4921 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4981 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5041 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5101 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5161 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5221 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5281 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5341 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5401 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5461 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5521 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5581 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6241 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6901 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7561 cgaaaagtgc cacctgacgt c
//
