LOCUS       sequence-180665-        8083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-HA-AGO2 [S824E, S828E, T830E, S831E, S834E (5xE)].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8083)
  AUTHORS   Golden RJ, Chen B, Li T, Braun J, Manjunath H, Chen X, Wu J, Schmid 
            V, Chang TC, Kopp F, Ramirez-Martinez A, Tagliabracci VS, Chen ZJ, 
            Xie Y, Mendell JT
  TITLE     An Argonaute phosphorylation cycle promotes microRNA-mediated 
            silencing.
  JOURNAL   Nature. 2017 Feb 9;542(7640):197-202. doi: 10.1038/nature21025. Epub
            2017 Jan 23.
  PUBMED    28114302
REFERENCE   2  (bases 1 to 8083)
  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: 
            "10.1038/nature21025"; journalName: "Nature"; date: "2017-02-9- 9"; 
            volume: "542"; issue: "7640"; pages: "197-202"
COMMENT     Sequence Label: pcDNA3.1+_FH-AGO2-5xE
FEATURES             Location/Qualifiers
     source          1..8083
                     /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             964..987
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             988..1014
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2950..2959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3674..3691)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3680..3904
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3950..4378
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4037..4056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4247..4268
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4387..4407)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4392..4721
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4572..4707
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4634..4653
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4788..5582
                     /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(4842..4861)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5452..5471
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5756..5877
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5793..5812)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5847..5866
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5926..5942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5926..5942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5939..5961)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5950..5966
                     /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(5974..5980,5981..5998,5999..6004))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6019..6040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6157..6174)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6328..6916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6408..6427)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7087..7878,7879..7947))
                     /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     7710..7729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7948..8052)
                     /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 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 atggactaca aggacgacga tgacaagtac ccttatgacg tgcccgatta cgctagcggc
     1021 cgcatgtact cgggagccgg ccccgcactt gcacctcctg cgccgccgcc ccccatccaa
     1081 ggatatgcct tcaagcctcc acctagaccc gactttggga cctccgggag aacaatcaaa
     1141 ttacaggcca atttcttcga aatggacatc cccaaaattg acatctatca ttatgaattg
     1201 gatatcaagc cagagaagtg cccgaggaga gttaacaggg aaatcgtgga acacatggtc
     1261 cagcacttta aaacacagat ctttggggat cggaagcccg tgtttgacgg caggaagaat
     1321 ctatacacag ccatgcccct tccgattggg agggacaagg tggagctgga ggtcacgctg
     1381 ccaggagaag gcaaggatcg catcttcaag gtgtccatca agtgggtgtc ctgcgtgagc
     1441 ttgcaggcgt tacacgatgc actttcaggg cggctgccca gcgtcccttt tgagacgatc
     1501 caggccctgg acgtggtcat gaggcacttg ccatccatga ggtacacccc cgtgggccgc
     1561 tccttcttca ccgcgtccga aggctgctct aaccctcttg gcgggggccg agaagtgtgg
     1621 tttggcttcc atcagtccgt ccggccttct ctctggaaaa tgatgctgaa tattgatgtg
     1681 tcagcaacag cgttttacaa ggcacagcca gtaatcgagt ttgtttgtga agttttggat
     1741 tttaaaagta ttgaagaaca acaaaaacct ctgacagatt cccaaagggt aaagtttacc
     1801 aaagaaatta aaggtctaaa ggtggagata acgcactgtg ggcagatgaa gaggaagtac
     1861 cgtgtctgca atgtgacccg gcggcccgcc agtcaccaaa cattcccgct gcagcaggag
     1921 agcgggcaga cggtggagtg cacggtggcc cagtatttca aggacaggca caagttggtt
     1981 ctgcgctacc cccacctccc atgtttacaa gtcggacagg agcagaaaca cacctacctt
     2041 cccctggagg tctgtaacat tgtggcagga caaagatgta ttaaaaaatt aacggacaat
     2101 cagacctcaa ccatgatcag agcgactgct aggtcggcgc ccgatcggca agaagagatt
     2161 agcaaattga tgcgaagtgc aagtttcaac acagatccat acgtccgtga atttggaatc
     2221 atggtcaaag atgagatgac agacgtgact gggcgggtgc tgcagccgcc ctccatcctc
     2281 tacgggggca ggaataaagc tattgcgacc cctgtccagg gcgtctggga catgcggaac
     2341 aagcagttcc acacgggcat cgagatcaag gtgtgggcca ttgcgtgctt cgccccccag
     2401 cgccagtgca cggaagtcca tctgaagtcc ttcacagagc agctcagaaa gatctcgaga
     2461 gacgccggca tgcccatcca gggccagccg tgcttctgca aatacgcgca gggggcggac
     2521 agcgtggagc ccatgttccg gcacctgaag aacacgtatg cgggcctgca gctggtggtg
     2581 gtcatcctgc ccggcaagac gcccgtgtac gccgaggtca agcgcgtggg agacacggtg
     2641 ctggggatgg ccacgcagtg cgtgcagatg aagaacgtgc agaggaccac gccacagacc
     2701 ctgtccaacc tctgcctgaa gatcaacgtc aagctgggag gcgtgaacaa catcctgctg
     2761 ccccagggca ggccgccggt gttccagcag cccgtcatct ttctgggagc agacgtcact
     2821 cacccccccg ccggggatgg gaagaagccc tccattgccg ccgtggtggg cagcatggac
     2881 gcccacccca atcgctactg cgccaccgtg cgcgtgcagc agcaccggca ggagatcata
     2941 caagacctgg ccgccatggt ccgcgagctc ctcatccagt tctacaagtc cacgcgcttc
     3001 aagcccaccc gcatcatctt ctaccgcgac ggtgtctctg aaggccagtt ccagcaggtt
     3061 ctccaccacg agttgctggc catccgtgag gcctgtatca agctagaaaa agactaccag
     3121 cccgggatca ccttcatcgt ggtgcagaag aggcaccaca cccggctctt ctgcactgac
     3181 aagaacgagc gggttgggaa aagtggaaac attccagcag gcacgactgt ggacacgaaa
     3241 atcacccacc ccaccgagtt cgacttctac ctgtgtagtc acgctggcat ccaggggaca
     3301 agcaggcctt cgcactatca cgtcctctgg gacgacaatc gtttctcctc tgatgagctg
     3361 cagatcctaa cctaccagct gtgtcacacc tacgtgcgct gcacacgctc cgtgtccatc
     3421 ccagcgccag catactacgc tcacctggtg gccttccggg ccaggtacca cctggtggat
     3481 aaggaacatg acgaggctga aggagagcat gaggaggggc aggagaacgg gcgagaccac
     3541 caagcactgg ccaaggcggt ccaggttcac caagacactc tgcgcaccat gtactttgct
     3601 tgagaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa
     3661 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3721 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3781 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3841 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3901 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3961 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4021 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4081 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4141 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4201 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4261 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4321 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     4381 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4441 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     4501 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4561 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4621 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     4681 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4741 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4801 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4861 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4921 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4981 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5041 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5101 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5161 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5221 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5281 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5341 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5401 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5461 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5521 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5581 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5641 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5701 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5761 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5821 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5881 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5941 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     6001 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     6061 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6121 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     6181 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     6241 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     6301 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6361 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6421 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6481 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6541 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6601 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6661 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6721 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6781 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6841 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6901 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6961 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7021 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7081 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7141 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7201 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7261 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7321 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7381 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7441 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7501 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7561 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7621 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7681 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7741 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7801 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7861 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7921 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7981 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8041 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
