LOCUS       sequence-443620-        7038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of TNRC6A-CIM1 domain with S1332A/S1346A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7038)
  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 7038)
  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-CIM1-S1332A/S1346A
FEATURES             Location/Qualifiers
     source          1..7038
                     /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(2629..2646)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2635..2859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2905..3333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2992..3011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3202..3223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3342..3362)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3347..3676
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3527..3662
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3589..3608
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3743..4537
                     /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(3797..3816)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4407..4426
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4711..4832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4748..4767)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4802..4821
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4881..4897)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4881..4897)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4894..4916)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4905..4921
                     /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(4929..4935,4936..4953,4954..4959))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4974..4995
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5112..5129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5283..5871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5363..5382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6042..6833,6834..6902))
                     /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     6665..6684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6903..7007)
                     /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 aaattgatct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     2641 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2701 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2761 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2821 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     2881 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2941 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3001 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3061 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3121 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3181 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3241 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3301 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3361 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3421 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3481 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3541 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3601 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3661 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3721 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3781 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3841 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3901 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3961 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4021 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4081 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4141 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4201 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4261 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4321 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4381 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4441 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4501 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4561 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4621 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4681 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4741 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4801 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4861 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4921 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4981 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5041 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5101 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5161 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5221 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5281 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5341 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5401 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5461 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5521 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5581 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5641 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5701 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5761 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5821 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5881 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5941 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6001 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6061 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6121 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6181 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6241 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6301 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6361 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6421 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6481 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6541 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6601 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6661 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6721 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6781 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6841 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6901 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6961 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7021 aaagtgccac ctgacgtc
//
