LOCUS       sequence-457984-        8371 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express mutant human AGO2 with N-terminal 3xFLAG-SUMO_Eu1 tag in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8371)
  AUTHORS   Mohamed AA, Wang PY, Bartel DP, Vos SM
  TITLE     The structural basis for RNA slicing by human Argonaute2.
  JOURNAL   Cell Rep. 2025 Jan 28;44(1):115166. doi: 
            10.1016/j.celrep.2024.115166. Epub 2024 Dec 31.
  PUBMED    39932188
REFERENCE   2  (bases 1 to 8371)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2025-01-28"; volume: "44(1):115166. doi"; pages: " 
            10.1016/j.celrep.2024.115166. Epub 2024 Dec 31"
COMMENT     Sequence Label: pcDNA3.3_3xFLAG_SUMO_Eu1_HsAGO2(H56A, R68A, R9...
FEATURES             Location/Qualifiers
     source          1..8371
                     /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      3052..3061
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3757..3776)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3801..3849
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4051..4479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4138..4157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4348..4369
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4488..4508)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4493..4822
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4673..4808
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4735..4754
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4889..5683
                     /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(4943..4962)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5553..5572
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5859..5980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5896..5915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5950..5969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6029..6045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6029..6045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6042..6064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6053..6069
                     /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(6077..6083,6084..6101,6102..6107))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6122..6143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6260..6277)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6431..7019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6511..6530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7190..7981,7982..8050))
                     /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     7813..7832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8051..8155)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8230..8249)
                     /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 gtggtgctag cggatctgct
      841 gcgggtggcg aagaagataa gaaaccggca ggtggcgaag gtggcggtgc ccatatcaac
      901 ctgaaagtga aaggtcaaga cggcaacgaa gtctttttcc gcatcaaacg ttctacccag
      961 ctgaaaaagc tgatgaacgc atactgtgac cgtcagtctg tagacatgaa ggcaattgct
     1021 ttcctcttta agggtcgtcg cctacgtgcg gaaaggaccc cggatgaact ggaaatggaa
     1081 gatggcgacg aaatcgacgc aatgctgcac cagactggag gcggatccta ctcgggagcc
     1141 ggccccgcac ttgcacctcc tgcgccgccg ccccccatcc aaggatatgc cttcaagcct
     1201 ccacctagac ccgactttgg gacctccggg agaacaatca aattacaggc caatttcttc
     1261 gaaatggaca tccccaaaat tgacatctat gcttatgaat tggatatcaa gccagagaag
     1321 tgcccggcca gagttaacag ggaaatcgtg gaacacatgg tccagcactt taaaacacag
     1381 atctttgggg atcggaagcc cgtgtttgac ggcgcggcga atctatacac agccatgccc
     1441 cttccgattg ggagggacaa ggtggagctg gaggtcacgc tgccaggaga aggcaaggat
     1501 cgcatcttca aggtgtccat caagtgggtg tcctgcgtga gcttgcaggc gttacacgat
     1561 gcactttcag ggcggctgcc tagcgtccct tttgagacga tccaggccct ggacgtggtc
     1621 atgaggcact tgccatccat gaggtacacc cccgtgggcc gctccttctt caccgcgtcc
     1681 gaaggctgct ctaaccctct tggcgggggc cgagaagtgt ggtttggctt ccatcagtcc
     1741 gtccggcctt ctctctggaa aatgatgctg aatattgatg tgtcagcaac agcgttttac
     1801 aaggcacagc cagtaatcga gtttgtttgt gaagttttgg attttaaaag tattgaagaa
     1861 caacaaaaac ctctgacaga ttcccaaagg gtaaagttta ccaaagaaat taaaggtcta
     1921 aaggtggaga taacgcactg tgggcagatg aagaggaagt accgcgtctg caatgtgacc
     1981 cggcggcccg ccagtcacca aacattcccg ctgcagcagg agagcgggca gacggtggag
     2041 tgcacggtgg cccagtattt caaggacagg cacaagcttg ttctgcgcta cccccacctc
     2101 ccatgtttac aagtcggaca ggagcagaaa cacacctacc ttcccctgga ggtctgtaac
     2161 attgtggcag gacaaagatg tattaaaaaa ttaacggaca atcagacctc aaccatgatc
     2221 agagcaactg ctaggtcggc gcccgatcgg caagaagaga ttagcaaatt gatgcgaagt
     2281 gcaagtttca acacagatcc atacgtccgt gaatttggaa tcatggtcaa agatgagatg
     2341 acagacgtga ctgggcgggt gctgcagccg ccctccatcc tctacggggg caggaataaa
     2401 gctattgcga cccctgtcca gggcgtctgg gacatgcgga acaagcagtt ccacacgggc
     2461 atcgagatca aggtgtgggc cattgcgtgc ttcgcccccc agcgccagtg cacggaagtc
     2521 catctgaagt ccttcacaga gcagctcaga aagatctcga gagacgctgg catgcccatc
     2581 cagggccagc cgtgcttctg caaatacgcg cagggggcgg acagcgtgga gcccatgttc
     2641 cggcacctga agaacacgta tgcgggcctg cagctggtgg tggtcatcct gcccggcaag
     2701 acgcccgtgt acgccgaggt caagcgcgtg ggagacacgg tgctggggat ggccacgcag
     2761 tgcgtgcaga tgaagaacgt gcagaggacc acgccacaga ccctgtccaa cctctgcctg
     2821 aagatcaacg tcaagctggg aggcgtgaac aacatcctgc tgccccaggg caggccgccg
     2881 gtgttccagc agcccgtcat ctttctggga gcagacgtca ctcacccccc cgccggggat
     2941 gggaagaagc cctccattgc cgccgtggtg ggcagcatgg acgcccaccc caatcgctac
     3001 tgcgccaccg tgcgcgtgca gcagcaccgg caggagatca tacaagacct ggccgccatg
     3061 gtccgcgagc tcctcatcca gttctacaag tccacgcgct tcaagcccac ccgcatcatc
     3121 ttctaccgcg acggtgtctc tgaaggccag ttccagcagg ttctccacca cgagttgctg
     3181 gccatccgtg aggcctgtat caagctagaa aaagactacc agcccgggat caccttcatc
     3241 gtggtgcaga agaggcacca cacccggctc ttctgcactg acaagaacga gcgggttggg
     3301 aaaagtggaa acattccagc aggcacgact gtggacacga aaatcaccca ccccaccgag
     3361 ttcgacttct acctgtgtag tcacgctggc atccagggga caagcaggcc ttcgcactat
     3421 cacgtcctct gggacgacaa tcgtttctcc tctgatgagc tgcagatcct aacctaccag
     3481 ctgtgtcaca cctacgtgcg ctgcacacgc tccgtgtcca tcccagcgcc agcatactac
     3541 gctcacctgg tggccttccg ggccaggtac cacctggtgg ataaggaaca tgacagtgct
     3601 gaaggaagcc atacctctgg gcagagtaac gggcgagacc accaagcact ggccaaggcg
     3661 gtccaggttc accaagacac tctgcgcacc atgtactttg cttgaaaggg ttcgatccct
     3721 accggttagt aatgagttta aacgggggag gctaactgaa acacggaagg agacaatacc
     3781 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt
     3841 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     3901 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     3961 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcagatc
     4021 tgcgcagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     4081 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4141 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4201 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4261 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4321 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4381 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4441 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4501 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4561 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4621 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4681 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4741 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     4801 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     4861 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4921 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4981 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     5041 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     5101 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     5161 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     5221 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     5281 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5341 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5401 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5461 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5521 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5581 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5641 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     5701 gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5761 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5821 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5941 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     6001 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     6061 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6121 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6181 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6241 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6301 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6361 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6421 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6481 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6541 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6601 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6661 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6721 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6781 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6841 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6901 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6961 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7021 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7081 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7141 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7201 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7261 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7321 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7381 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7441 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7501 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7561 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7621 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7681 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7741 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7801 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7861 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7921 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7981 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8041 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8101 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8161 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     8221 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     8281 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     8341 gaccgacaat tgcatgaaga atctgcttag g
//
