LOCUS       sequence-436012-        8071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express S387 phosphomimetic mutant human AGO2 with N-terminal 3xFLAG
            tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8071)
  AUTHORS   Wang PY, Bartel DP
  TITLE     The guide-RNA sequence dictates the slicing kinetics and 
            conformational dynamics of the Argonaute silencing complex.
  JOURNAL   Mol Cell. 2024 Jul 10:S1097-2765(24)00533-1. doi: 
            10.1016/j.molcel.2024.06.026.
  PUBMED    39025072
REFERENCE   2  (bases 1 to 8071)
  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: "Mol Cell. 
            2024 Jul 10:S1097-2765(24)00533-1. doi: 
            10.1016/j.molcel.2024.06.026."
COMMENT     Sequence Label: pcDNA3.3−3xFLAG−AGO2(S387D)
FEATURES             Location/Qualifiers
     source          1..8071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             751..816
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2752..2761
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3457..3476)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3501..3549
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3751..4179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3838..3857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4048..4069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4188..4208)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4193..4522
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4373..4508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4435..4454
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4589..5383
                     /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(4643..4662)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5253..5272
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5559..5680
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5596..5615)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5650..5669
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5729..5745)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5729..5745)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5742..5764)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5753..5769
                     /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(5777..5783,5784..5801,5802..5807))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5822..5843
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5960..5977)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6131..6719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6211..6230)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6890..7681,7682..7750))
                     /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     7513..7532
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7751..7855)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7930..7949)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gactacaaag accatgacgg tgattataaa
      781 gatcatgata tcgattacaa ggatgacgat gacaagcttg gtggttctta ctcgggagcc
      841 ggccccgcac ttgcacctcc tgcgccgccg ccccccatcc aaggatatgc cttcaagcct
      901 ccacctagac ccgactttgg gacctccggg agaacaatca aattacaggc caatttcttc
      961 gaaatggaca tccccaaaat tgacatctat cattatgaat tggatatcaa gccagagaag
     1021 tgcccgagga gagttaacag ggaaatcgtg gaacacatgg tccagcactt taaaacacag
     1081 atctttgggg atcggaagcc cgtgtttgac ggcaggaaga atctatacac agccatgccc
     1141 cttccgattg ggagggacaa ggtggagctg gaggtcacgc tgccaggaga aggcaaggat
     1201 cgcatcttca aggtgtccat caagtgggtg tcctgcgtga gcttgcaggc gttacacgat
     1261 gcactttcag ggcggctgcc tagcgtccct tttgagacga tccaggccct ggacgtggtc
     1321 atgaggcact tgccatccat gaggtacacc cccgtgggcc gctccttctt caccgcgtcc
     1381 gaaggctgct ctaaccctct tggcgggggc cgagaagtgt ggtttggctt ccatcagtcc
     1441 gtccggcctt ctctctggaa aatgatgctg aatattgatg tgtcagcaac agcgttttac
     1501 aaggcacagc cagtaatcga gtttgtttgt gaagttttgg attttaaaag tattgaagaa
     1561 caacaaaaac ctctgacaga ttcccaaagg gtaaagttta ccaaagaaat taaaggtcta
     1621 aaggtggaga taacgcactg tgggcagatg aagaggaagt accgcgtctg caatgtgacc
     1681 cggcggcccg ccagtcacca aacattcccg ctgcagcagg agagcgggca gacggtggag
     1741 tgcacggtgg cccagtattt caaggacagg cacaagcttg ttctgcgcta cccccacctc
     1801 ccatgtttac aagtcggaca ggagcagaaa cacacctacc ttcccctgga ggtctgtaac
     1861 attgtggcag gacaaagatg tattaaaaaa ttaacggaca atcagacctc aaccatgatc
     1921 agagcaactg ctaggtcggc gcccgatcgg caagaagaga ttagcaaatt gatgcgaagt
     1981 gcagatttca acacagatcc atacgtccgt gaatttggaa tcatggtcaa agatgagatg
     2041 acagacgtga ctgggcgggt gctgcagccg ccctccatcc tctacggggg caggaataaa
     2101 gctattgcga cccctgtcca gggcgtctgg gacatgcgga acaagcagtt ccacacgggc
     2161 atcgagatca aggtgtgggc cattgcgtgc ttcgcccccc agcgccagtg cacggaagtc
     2221 catctgaagt ccttcacaga gcagctcaga aagatctcga gagacgctgg catgcccatc
     2281 cagggccagc cgtgcttctg caaatacgcg cagggggcgg acagcgtgga gcccatgttc
     2341 cggcacctga agaacacgta tgcgggcctg cagctggtgg tggtcatcct gcccggcaag
     2401 acgcccgtgt acgccgaggt caagcgcgtg ggagacacgg tgctggggat ggccacgcag
     2461 tgcgtgcaga tgaagaacgt gcagaggacc acgccacaga ccctgtccaa cctctgcctg
     2521 aagatcaacg tcaagctggg aggcgtgaac aacatcctgc tgccccaggg caggccgccg
     2581 gtgttccagc agcccgtcat ctttctggga gcagacgtca ctcacccccc cgccggggat
     2641 gggaagaagc cctccattgc cgccgtggtg ggcagcatgg acgcccaccc caatcgctac
     2701 tgcgccaccg tgcgcgtgca gcagcaccgg caggagatca tacaagacct ggccgccatg
     2761 gtccgcgagc tcctcatcca gttctacaag tccacgcgct tcaagcccac ccgcatcatc
     2821 ttctaccgcg acggtgtctc tgaaggccag ttccagcagg ttctccacca cgagttgctg
     2881 gccatccgtg aggcctgtat caagctagaa aaagactacc agcccgggat caccttcatc
     2941 gtggtgcaga agaggcacca cacccggctc ttctgcactg acaagaacga gcgggttggg
     3001 aaaagtggaa acattccagc aggcacgact gtggacacga aaatcaccca ccccaccgag
     3061 ttcgacttct acctgtgtag tcacgctggc atccagggga caagcaggcc ttcgcactat
     3121 cacgtcctct gggacgacaa tcgtttctcc tctgatgagc tgcagatcct aacctaccag
     3181 ctgtgtcaca cctacgtgcg ctgcacacgc tccgtgtcca tcccagcgcc agcatactac
     3241 gctcacctgg tggccttccg ggccaggtac cacctggtgg ataaggaaca tgacagtgct
     3301 gaaggaagcc atacctctgg gcagagtaac gggcgagacc accaagcact ggccaaggcg
     3361 gtccaggttc accaagacac tctgcgcacc atgtactttg cttgaaaggg ttcgatccct
     3421 accggttagt aatgagttta aacgggggag gctaactgaa acacggaagg agacaatacc
     3481 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt
     3541 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     3601 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     3661 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcagatc
     3721 tgcgcagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3781 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3841 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3901 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3961 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4021 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4081 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4141 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4201 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4261 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4321 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4381 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4441 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     4501 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     4561 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4621 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4681 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4741 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4801 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4861 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4921 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4981 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5041 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5101 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5161 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5221 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5281 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5341 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     5401 gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5461 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5521 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5581 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5641 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5701 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5761 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5821 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5881 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5941 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6001 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6061 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6121 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6181 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6241 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6301 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6361 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6421 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6481 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6541 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6601 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6661 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6721 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6781 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6841 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6901 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6961 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7021 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7081 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7141 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7201 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7261 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7321 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7381 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7441 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7501 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7561 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7621 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7681 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7741 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7801 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7861 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7921 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7981 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     8041 gaccgacaat tgcatgaaga atctgcttag g
//
