LOCUS       sequence-430705-        7908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AGO2-Y529E mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7908)
  AUTHORS   Nishi K, Takahashi T, Suzawa M, Miyakawa T, Nagasawa T, Ming Y, 
            Tanokura M, Ui-Tei K
  TITLE     Control of the localization and function of a miRNA silencing 
            component TNRC6A by Argonaute protein.
  JOURNAL   Nucleic Acids Res. 2015 Nov 16;43(20):9856-73. doi: 
            10.1093/nar/gkv1026. Epub 2015 Oct 7.
  PUBMED    26446993
REFERENCE   2  (bases 1 to 7908)
  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: 
            "10.1093/nar/gkv1026"; journalName: "Nucleic Acids Res"; date: 
            "2015-11-16- 16"; volume: "43"; issue: "20"; pages: "9856-73"
COMMENT     Sequence Label: pIRESneo-FLAG/HA-Ago2-Y529E
FEATURES             Location/Qualifiers
     source          1..7908
                     /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"
     regulatory      922..931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             931..954
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             955..981
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2917..2926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          3649..3878
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3861..3880
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3951..4525,4526..4528,4529..4537)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4118..4135)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4345..4364
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4560..5363
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4623..4642)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5233..5252
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5395..5412)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5401..5625
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5672..5691
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5751..5767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5751..5767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5764..5786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5775..5791
                     /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(5799..5805,5806..5823,5824..5829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5844..5865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5982..5999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6153..6741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6233..6252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6912..7703,7704..7772))
                     /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     7535..7554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7773..7877)
                     /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 gcttggtacc
      901 gagctcggat cgatatcgcc ggccgccatg gactacaagg acgacgatga caagtaccct
      961 tatgacgtgc ccgattacgc tagcggccgc atgtactcgg gagccggccc cgcacttgca
     1021 cctcctgcgc cgccgccccc catccaagga tatgccttca agcctccacc tagacccgac
     1081 tttgggacct ccgggagaac aatcaaatta caggccaatt tcttcgaaat ggacatcccc
     1141 aaaattgaca tctatcatta tgaattggat atcaagccag agaagtgccc gaggagagtt
     1201 aacagggaaa tcgtggaaca catggtccag cactttaaaa cacagatctt tggggatcgg
     1261 aagcccgtgt ttgacggcag gaagaatcta tacacagcca tgccccttcc gattgggagg
     1321 gacaaggtgg agctggaggt cacgctgcca ggagaaggca aggatcgcat cttcaaggtg
     1381 tccatcaagt gggtgtcctg cgtgagcttg caggcgttac acgatgcact ttcagggcgg
     1441 ctgcccagcg tcccttttga gacgatccag gccctggacg tggtcatgag gcacttgcca
     1501 tccatgaggt acacccccgt gggccgctcc ttcttcaccg cgtccgaagg ctgctctaac
     1561 cctcttggcg ggggccgaga agtgtggttt ggcttccatc agtccgtccg gccttctctc
     1621 tggaaaatga tgctgaatat tgatgtgtca gcaacagcgt tttacaaggc acagccagta
     1681 atcgagtttg tttgtgaagt tttggatttt aaaagtattg aagaacaaca aaaacctctg
     1741 acagattccc aaagggtaaa gtttaccaaa gaaattaaag gtctaaaggt ggagataacg
     1801 cactgtgggc agatgaagag gaagtaccgt gtctgcaatg tgacccggcg gcccgccagt
     1861 caccaaacat tcccgctgca gcaggagagc gggcagacgg tggagtgcac ggtggcccag
     1921 tatttcaagg acaggcacaa gttggttctg cgctaccccc acctcccatg tttacaagtc
     1981 ggacaggagc agaaacacac ctaccttccc ctggaggtct gtaacattgt ggcaggacaa
     2041 agatgtatta aaaaattaac ggacaatcag acctcaacca tgatcagagc gactgctagg
     2101 tcggcgcccg atcggcaaga agagattagc aaattgatgc gaagtgcaag tttcaacaca
     2161 gatccatacg tccgtgaatt tggaatcatg gtcaaagatg agatgacaga cgtgactggg
     2221 cgggtgctgc agccgccctc catcctctac gggggcagga ataaagctat tgcgacccct
     2281 gtccagggcg tctgggacat gcggaacaag cagttccaca cgggcatcga gatcaaggtg
     2341 tgggccattg cgtgcttcgc cccccagcgc cagtgcacgg aagtccatct gaagtccttc
     2401 acagagcagc tcagaaagat ctcgagagac gccggcatgc ccatccaggg ccagccgtgc
     2461 ttctgcaaat acgcgcaggg ggcggacagc gtggagccca tgttccggca cctgaagaac
     2521 acgtatgcgg gcctgcagct ggtggtggtc atcctgcccg gcaagacgcc cgtggaagcc
     2581 gaggtcaagc gcgtgggaga cacggtgctg gggatggcca cgcagtgcgt gcagatgaag
     2641 aacgtgcaga ggaccacgcc acagaccctg tccaacctct gcctgaagat caacgtcaag
     2701 ctgggaggcg tgaacaacat cctgctgccc cagggcaggc cgccggtgtt ccagcagccc
     2761 gtcatctttc tgggagcaga cgtcactcac ccccccgccg gggatgggaa gaagccctcc
     2821 attgccgccg tggtgggcag catggacgcc caccccaatc gctactgcgc caccgtgcgc
     2881 gtgcagcagc accggcagga gatcatacaa gacctggccg ccatggtccg cgagctcctc
     2941 atccagttct acaagtccac gcgcttcaag cccacccgca tcatcttcta ccgcgacggt
     3001 gtctctgaag gccagttcca gcaggttctc caccacgagt tgctggccat ccgtgaggcc
     3061 tgtatcaagc tagaaaaaga ctaccagccc gggatcacct tcatcgtggt gcagaagagg
     3121 caccacaccc ggctcttctg cactgacaag aacgagcggg ttgggaaaag tggaaacatt
     3181 ccagcaggca cgactgtgga cacgaaaatc acccacccca ccgagttcga cttctacctg
     3241 tgtagtcacg ctggcatcca ggggacaagc aggccttcgc actatcacgt cctctgggac
     3301 gacaatcgtt tctcctctga tgagctgcag atcctaacct accagctgtg tcacacctac
     3361 gtgcgctgca cacgctccgt gtccatccca gcgccagcat actacgctca cctggtggcc
     3421 ttccgggcca ggtaccacct ggtggataag gaacatgaca gtgctgaagg aagccatacc
     3481 tctgggcaga gtaacgggcg agaccaccaa gcactggcca aggcggtcca ggttcaccaa
     3541 gacactctgc gcaccatgta ctttgcttga cggaattcag tggatccact agtaacggcc
     3601 gccagtgtgc tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc
     3661 tctcaaaagc gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt
     3721 tgatattcac ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa
     3781 agacaatctt tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt
     3841 gagtgacaat gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact
     3901 gcaggtcgag catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctctccc
     3961 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     4021 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     4081 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     4141 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     4201 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     4261 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     4321 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     4381 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     4441 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     4501 ttcctttgaa aaacacgatg ataagcttgc cacaacccgg gataattcct gcagccaata
     4561 tgggatcggc cattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4621 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4681 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4741 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4801 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4861 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4921 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4981 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5041 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     5101 tgcccgacgg cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5161 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5221 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5281 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5341 gccttcttga cgagttcttc tgaggggatc aattctctag agctcgctga tcagcctcga
     5401 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5461 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5521 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5581 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5641 gaaccagctg gggctcgagt gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5701 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5761 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5821 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5881 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5941 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6001 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6061 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6121 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6181 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6241 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6301 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6361 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6421 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6481 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6541 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6601 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6661 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6721 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6781 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6841 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6901 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6961 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7021 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7081 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7141 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7201 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7261 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7321 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7381 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7441 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7501 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7561 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7621 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7681 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7741 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7801 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7861 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
