LOCUS       sequence-75861-e        8543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FLAG tagged Ago1 in S2 cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8543)
  AUTHORS   Kawamata T, Seitz H, Tomari Y
  TITLE     Structural determinants of miRNAs for RISC loading and 
            slicer-independent unwinding.
  JOURNAL   Nat Struct Mol Biol. 2009 Sep;16(9):953-60. doi: 10.1038/nsmb.1630. 
            Epub 2009 Aug 16.
  PUBMED    19684602
REFERENCE   2  (bases 1 to 8543)
  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/nsmb.1630";
            journalName: "Nat Struct Mol Biol"; date: "2009-09"; volume: "16"; 
            issue: "9"; pages: "953-60"
COMMENT     Sequence Label: pAFW-Ago1
FEATURES             Location/Qualifiers
     source          1..8543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        139..2648
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      2757..2766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2766..2831
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    2841..2865
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5755..5779)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5871..5936
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             6066..6086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6511..6645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6548..6567)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6602..6621
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6742..6810,6811..7602)
                     /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     complement(6960..6979)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7773..8361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8262..8281
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8515..8532
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg caggcaactc
       61 gtgaaaggta ggcggatcag cggccgcagc atgcaattct atattctaaa aacacaaatg
      121 atacttctaa aaaaaaatca tgaatggcat caactctgaa tcaaatcttt gcagatgcac
      181 ctacttctca tttccactgt cacatcattt ttccagatct cgctgcctgt tatgtggccc
      241 acaaaccaag acacgtttta tggccattaa agctggctga tcgtcgccaa acaccaaata
      301 cataatgaat atgtacacat tcgagaaaga agcgatcaaa gaagcgtctt cgggcggagt
      361 aggagaatgc ggaggagaag gagaacgagc tgatctagta tctctccaca atccaatgcc
      421 aactgaccaa ctggccatat tcggagcaat ttgaagccaa tttccatcgc ctggcgatcg
      481 ctccattctt ggctatatgt ttttcaccgt tacccggggc cattttcaaa gactcgtcgg
      541 caagataaga ttgtgtcact cgctgtctct cttcatttgt cgaagaatgc tgaggaattt
      601 cgcgatgacg tcggcgagta ttttgaagaa tgagaataat ttgtatttat acgaaaatca
      661 gttagtggaa ttttctacaa aaacatgtta tctatagata attttgttgc aaaatatgtt
      721 gactatgaca aagattgtat gtatatacct ttaatgtatt ctcattttct tatgtattta
      781 taatggcaat gatgatactg atgatatttt aagatgatgc cagaccaaaa ggcttgaatt
      841 tctgcgtctt ttgccgaacg cagtgcatgt gcaattgttg ttttttggaa tattcaattt
      901 tcggactgtc cgctttgatt tcagtttctt ggcttattca aaaagcaaag taaagccaaa
      961 aaagcgagat ggcaatacca aatgcggcaa aacggtagtg gaaggaaagg ggtgcggggc
     1021 agcggaagga agggtggggc ggggcgtggc ggggtctgtg gctgggcgcg acgtcaccga
     1081 cgttggagcc actcctttga ccatgtgtgc gtgtgtgtat tattcgtgtc tcgccactcg
     1141 ccggttgttt ttttcttttt atgctgcgct ctctctagcg ccatctcgct tacgcatgct
     1201 caacgcaccg catgttgccg tttcctttta tgcgtcattt tggctcgaaa taggcaatta
     1261 tttaaacaaa gattagtcaa cgaaaacgct aaaataaata agtctacaat atggttactt
     1321 attgccatgt gtgtgcagcc aacgatagca acaaaagcaa caacacaggt ggctttccct
     1381 ctttcacttt ttgtttgcaa gccgcgtgcg agcaagacgg cacgaccggc aaacgcaatt
     1441 acgctgacaa agagcagacg aagttttggc gaaaaacatc aaggcgcctg atacgaatgc
     1501 atttgcaata acaattgcga tatttaatat tgtttatgaa gctgtttgac ttcaaaacac
     1561 acaaaaaaaa aaataaaaca aattatttga aagagaatta ggaatcggac gcttatcgtt
     1621 agggtaacaa caagaaatgc ttactgagtc acagcctctg gaaaactgcc gcaagccaga
     1681 gagagagaga aaaagaggga gagcagctta gaccgcatgt gcttgtgtgt gaggcgtctc
     1741 tctcttcgtc tctgttgcgc aaacgcatag actgcactga gaaaatcgat tacctatttt
     1801 ttatgaatga atatttgcac tattactatt caaaactatt aagatagcaa tcacattcaa
     1861 tagccaaata ctataccacc tgagcgatgc aacgaaatga tcaatttgag caaaaatgct
     1921 gcatatttag gacggcatca ttatagaaat gcttcttgct gtgtactttt ctctcgtctg
     1981 gcagctgttt cgccgttatt gttaaaaccg gcttaagtta ggtgtgtttt ctacgactag
     2041 tgaatgccct actagaagat gtgtgttgca caaaatgtcc ctggaataac caatttgaag
     2101 tgcagatagc agtaaacgta agctaatatg aatattattt aactgtaatg ttttaatatc
     2161 gctggacatt actaataaac ccactataaa cacatgtaca tatgtatgtt ttggcataca
     2221 atgagtagtt ggggaaaaaa tgtgtaaaag caccgtgacc atcacagcat aaagataacc
     2281 agctgaagta tcgaatatga gtaaccccca aattgaatca catgccgcaa ctgataggac
     2341 ccatggaagt acactcttca tggcgatata caagacacac acaagcacga acacccagtt
     2401 gcggaggaaa ttctccgtaa atgaaaaccc aatcggcgaa caattcatac ccatatatgg
     2461 taaaagtttt gaacgcgact tgagagcgga gagcattgcg gctgataagg ttttagcgct
     2521 aagcgggctt tataaaacgg gctgcgggac cagttttcat atcactaccg tttgagttct
     2581 tgtgctgtgt ggatactcct cccgacacaa agccgctcca tcagccagca gtcgtctaat
     2641 ccagagaccc cggatccaga tatcgaggcc tgtctagaga agcttgttcg aatctcgagt
     2701 gcgcgcttcc ggaggtatac acctaggcgg taccactgca gtgaattcgg agctccgcca
     2761 ccatggacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     2821 acgatgacaa gcaccggtat acaagtttgt acaaaaaagc aggctccgcg gccgccccct
     2881 tcaccatgta tccagttgga caacagtcac agtggacccc ctcgcccact cggcctcaga
     2941 gtccctctca ggcgcagact agcttcgaca cgctcacatc accaccagcg cccggctcgt
     3001 cggtcaatcc caccgccgtc accagcccaa gtgcccagaa tgtggccgct ggtggagcaa
     3061 ctgtggccgg tgcagcagca actgccgccc aggtggcctc cgccttgggt gccaccaccg
     3121 gtagcgtgac gccagcaatt gccaccgcaa cgccagccac gcagccggat atgcccgtct
     3181 ttacgtgtcc acgacgcccg aatctcggac gagagggtcg cccgattgtg ctgcgcgcca
     3241 atcacttcca ggtgacaatg ccgcgtggct atgtgcatca ctatgacatc aatatacagc
     3301 cggacaagtg tccgcgaaag gtgaaccgtg agattatcga gactatggtg catgcctata
     3361 gcaagatctt cggagtgctt aagcctgtgt tcgatggtcg caacaatctg tatacccgcg
     3421 atcccctgcc cattggcaac gagcgtttgg agctggaggt tactctaccc ggcgagggca
     3481 aagatcgaat ctttcgcgtg acgatcaagt ggcaggctca ggtctcgctc ttcaatttgg
     3541 aggaagctct cgaaggccgc acgcggcaga taccctatga tgccattttg gcgctcgatg
     3601 tggtcatgcg ccatctgccc agcatgacgt acacgccagt gggacgtagc ttcttcagtt
     3661 ccccggaggg ttactaccat cccctgggtg gtggacgcga ggtttggttc ggtttccatc
     3721 agagcgtaag gccctcgcag tggaagatga tgctcaatat cgatgtctcg gccaccgctt
     3781 tctacaaggc tcaaccagtc attgacttca tgtgcgaggt gctggacatt cgcgacatca
     3841 acgagcagcg caaaccgctc accgattcgc agcgcgtcaa gttcacgaag gagatcaagg
     3901 gtttgaagat cgagatcacc cactgcggcc agatgcgtcg caagtatcgt gtgtgcaacg
     3961 tcactcgccg ccccgctcag atgcaatcat tcccactgca gctggagaac ggacagaccg
     4021 tagagtgcac cgtggccaag tacttcctgg acaagtaccg catgaaattg cgctacccgc
     4081 acttgccctg cctgcaggtt ggccaagagc acaagcacac ttacctgcct ctagaggtgt
     4141 gcaacattgt ggccggacag cggtgcatta aaaagctgac cgatatgcag acgtcgacca
     4201 tgatcaaggc cacagctcgt tcagctccgg atcgtgagcg tgagattaac aacttggtaa
     4261 agcgcgccga cttcaacaac gattcgtatg tgcaagagtt tggcctgacc atctccaatt
     4321 cgatgatgga ggtacgagga cgcgtcttgc ctcctcccaa gcttcagtat gggggacgtg
     4381 tgtctaccgg cctcaccggc cagcagctgt tcccgccaca gaacaaggtg agcttggcct
     4441 cgcccaacca gggtgtatgg gatatgcgag gcaagcagtt cttcactggc gtcgagatcc
     4501 gcatctgggc catcgcctgt tttgccccac agcgcacggt gcgcgaggat gcgctgcgta
     4561 atttcaccca gcagctgcag aagatctcaa acgatgcagg catgccgata attggacagc
     4621 cgtgcttctg taagtacgcc accgggccgg atcaagtgga acccatgttc cgttacctga
     4681 agatcacctt ccccggcctg caactcgtcg tggttgtgct gcccggcaag actccagtat
     4741 acgccgaggt gaagcgtgta ggtgacaccg ttctgggtat ggccacccag tgtgtgcagg
     4801 ccaagaacgt gaacaagaca tcgccacaga cgctctctaa tctgtgtctg aagatcaacg
     4861 tcaagttggg cggcatcaat tcaattctgg tcccctccat tcggccaaag gtcttcaatg
     4921 agccggtcat ctttttgggt gccgatgtga cacacccacc agctggcgac aacaagaaac
     4981 catcgattgc cgccgtcgtg ggctccatgg atgcccatcc atcgcggtat gccgccaccg
     5041 ttcgcgtaca gcagcaccga caggagatca tccaggagct gagcagcatg gtgcgcgagc
     5101 tgttgatcat gttctacaag tcgacgggcg gctacaagcc ccaccgcatc atactctatc
     5161 gtgacggagt ctccgaggga caattcccac atgtcctgca acacgaattg accgccattc
     5221 gcgaggcctg cattaagcta gagccagagt atcggccggg catcacattc attgtggtgc
     5281 agaagcgcca tcacactcga ctcttctgcg cggagaagaa ggagcagagc ggcaaatcgg
     5341 gcaatattcc cgcaggcacc accgtcgatg tgggcatcac acatcccacc gaatttgatt
     5401 tctatctatg cagccatcag ggcatccagg gcaccagtcg cccctcgcac taccacgttc
     5461 tgtgggacga caatcacttt gactcggacg agctgcagtg cctcacgtat cagctatgcc
     5521 atacgtacgt gcgctgcacc cgatccgtca gtataccggc gccagcctac tacgcccatt
     5581 tagtggcctt ccgtgccaga tatcatctgg tggagaagga gcacgattcg ggcgagggtt
     5641 cgcaccagag cggctgctca gaggatcgta cgccaggtgc catggccagg gccatcactg
     5701 tgcacgcgga taccaagaag gtcatgtact ttgcctaaaa gggtgggcgc gccgacccag
     5761 ctttcttgta caaagtggtg acgtaagcta gcaggatctt tgtgaaggaa ccttacttct
     5821 gtggtgtgac ataattggac aaactaccta cagagattta aagctctaag gtaaatataa
     5881 aatttttaag tgtataatgt gttaaactac tgattctaat tgtttgtgta ttttagattc
     5941 caacctatgg aactgatgaa tgggagcagt ggtggaatgc ctttaatgag gaaaacctgt
     6001 tttgctcaga agaaatgcca tctagtgatg atgaggctac tgctgactct caacattcta
     6061 ctcctccaaa aaagaagaga aaggtagaag accccaagga ctttccttca gaattgctaa
     6121 gttttttgag tcatgctgtg tttagtaata gaactcttgc ttgctttgct atttacacca
     6181 caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct gtaaccttta
     6241 taagtaggca taacagttat aatcataaca tactgttttt tcttactcca cacaggcata
     6301 gagtgtctgc tattaataac tatgctcaaa aattgtgtac ctttagcttt ttaatttgta
     6361 aaggggttaa taaggaatat ttgatgtata gtgccttgac tagagatcat aatcagccat
     6421 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     6481 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     6541 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     6601 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcccgtt taaactacgc
     6661 gtaattcaaa cagggttctg gcgtcgttct cgtactgttt tccccaggcc agtgctttag
     6721 cgttattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6781 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6841 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6901 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6961 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7021 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7081 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     7141 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     7201 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     7261 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     7321 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7381 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7441 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7501 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7561 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7621 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7681 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7741 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7801 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7861 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     7921 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7981 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     8041 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8101 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     8161 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8221 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8281 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8341 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8401 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8461 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8521 tcagtgagcg aggaagcgga aga
//
