LOCUS       sequence-375838-        7115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flag-tagged Nibbler variants expression in Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  AUTHORS   Xie W, Sowemimo I, Hayashi R, Wang J, Burkard TR, Brennecke J, 
            Ameres SL, Patel DJ
  TITLE     Structure-function analysis of microRNA 3'-end trimming by Nibbler.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Dec 1;117(48):30370-30379. doi: 
            10.1073/pnas.2018156117. Epub 2020 Nov 16.
  PUBMED    33199607
REFERENCE   2  (bases 1 to 7115)
  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: 
            "10.1073/pnas.2018156117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-12-1- 1"; volume: "117"; issue: "48"; pages: 
            "30370-30379"
COMMENT     Sequence Label: pAFMW-Nbr[N1-383]
FEATURES             Location/Qualifiers
     source          1..7115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          106..171
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             301..321
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    746..880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(783..802)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     837..856
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(977..1045,1046..1837)
                     /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(1195..1214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2008..2596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2497..2516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2750..2767
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2918..5426
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      5535..5544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5544..5609
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      5625..5634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5634..5663
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5673..5702
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5712..5741
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5751..5780
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5790..5819
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5853..5882
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    5895..5919
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(join(7105..7115,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggtgacgta agctagcagg atctttgtga aggaacctta cttctgtggt
       61 gtgacataat tggacaaact acctacagag atttaaagct ctaaggtaaa tataaaattt
      121 ttaagtgtat aatgtgttaa actactgatt ctaattgttt gtgtatttta gattccaacc
      181 tatggaactg atgaatggga gcagtggtgg aatgccttta atgaggaaaa cctgttttgc
      241 tcagaagaaa tgccatctag tgatgatgag gctactgctg actctcaaca ttctactcct
      301 ccaaaaaaga agagaaaggt agaagacccc aaggactttc cttcagaatt gctaagtttt
      361 ttgagtcatg ctgtgtttag taatagaact cttgcttgct ttgctattta caccacaaag
      421 gaaaaagctg cactgctata caagaaaatt atggaaaaat attctgtaac ctttataagt
      481 aggcataaca gttataatca taacatactg ttttttctta ctccacacag gcatagagtg
      541 tctgctatta ataactatgc tcaaaaattg tgtaccttta gctttttaat ttgtaaaggg
      601 gttaataagg aatatttgat gtatagtgcc ttgactagag atcataatca gccataccac
      661 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
      721 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
      781 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      841 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc ccgtttaaac tacgcgtaat
      901 tcaaacaggg ttctggcgtc gttctcgtac tgttttcccc aggccagtgc tttagcgtta
      961 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1021 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1081 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1141 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1201 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1261 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1321 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1381 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1441 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1501 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     1561 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1621 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1681 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1741 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     1801 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1981 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2041 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2101 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2161 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2221 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2281 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2341 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2401 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2461 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2521 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2581 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2641 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2701 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2761 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2821 tcattaatgc aggcaactcg tgaaaggtag gcggatcagc ggccgcagca tgcaattcta
     2881 tattctaaaa acacaaatga tacttctaaa aaaaaatcat gaatggcatc aactctgaat
     2941 caaatctttg cagatgcacc tacttctcat ttccactgtc acatcatttt tccagatctc
     3001 gctgcctgtt atgtggccca caaaccaaga cacgttttat ggccattaaa gctggctgat
     3061 cgtcgccaaa caccaaatac ataatgaata tgtacacatt cgagaaagaa gcgatcaaag
     3121 aagcgtcttc gggcggagta ggagaatgcg gaggagaagg agaacgagct gatctagtat
     3181 ctctccacaa tccaatgcca actgaccaac tggccatatt cggagcaatt tgaagccaat
     3241 ttccatcgcc tggcgatcgc tccattcttg gctatatgtt tttcaccgtt acccggggcc
     3301 attttcaaag actcgtcggc aagataagat tgtgtcactc gctgtctctc ttcatttgtc
     3361 gaagaatgct gaggaatttc gcgatgacgt cggcgagtat tttgaagaat gagaataatt
     3421 tgtatttata cgaaaatcag ttagtggaat tttctacaaa aacatgttat ctatagataa
     3481 ttttgttgca aaatatgttg actatgacaa agattgtatg tatatacctt taatgtattc
     3541 tcattttctt atgtatttat aatggcaatg atgatactga tgatatttta agatgatgcc
     3601 agaccaaaag gcttgaattt ctgcgtcttt tgccgaacgc agtgcatgtg caattgttgt
     3661 tttttggaat attcaatttt cggactgtcc gctttgattt cagtttcttg gcttattcaa
     3721 aaagcaaagt aaagccaaaa aagcgagatg gcaataccaa atgcggcaaa acggtagtgg
     3781 aaggaaaggg gtgcggggca gcggaaggaa gggtggggcg gggcgtggcg gggtctgtgg
     3841 ctgggcgcga cgtcaccgac gttggagcca ctcctttgac catgtgtgcg tgtgtgtatt
     3901 attcgtgtct cgccactcgc cggttgtttt tttcttttta tgctgcgctc tctctagcgc
     3961 catctcgctt acgcatgctc aacgcaccgc atgttgccgt ttccttttat gcgtcatttt
     4021 ggctcgaaat aggcaattat ttaaacaaag attagtcaac gaaaacgcta aaataaataa
     4081 gtctacaata tggttactta ttgccatgtg tgtgcagcca acgatagcaa caaaagcaac
     4141 aacacaggtg gctttccctc tttcactttt tgtttgcaag ccgcgtgcga gcaagacggc
     4201 acgaccggca aacgcaatta cgctgacaaa gagcagacga agttttggcg aaaaacatca
     4261 aggcgcctga tacgaatgca tttgcaataa caattgcgat atttaatatt gtttatgaag
     4321 ctgtttgact tcaaaacaca caaaaaaaaa ataaaacaaa ttatttgaaa gagaattagg
     4381 aatcggacgc ttatcgttag ggtaacaaca agaaatgctt actgagtcac agcctctgga
     4441 aaactgccgc aagccagaga gagagagaaa aagagggaga gcagcttaga ccgcatgtgc
     4501 ttgtgtgtga ggcgtctctc tcttcgtctc tgttgcgcaa acgcatagac tgcactgaaa
     4561 aaatcgatta cctatttttt atgaatgaat atttgcacta ttactattca aaactattaa
     4621 gatagcaatc acattcaata gccaaatact ataccacctg agcgatgcaa cgaaatgatc
     4681 aatttgagca aaaatgctgc atatttagga cggcatcatt atagaaatgc ttcttgctgt
     4741 gtacttttct ctcgtctggc agctgtttcg ccgttattgt taaaaccggc ttaagttagg
     4801 tgtgttttct acgactagtg aatgccctac tagaagatgt gtgttgcaca aaatgtccct
     4861 ggaataacca atttgaagtg cagatagcag taaacgtaag ctaatatgaa tattatttaa
     4921 ctgtaatgtt ttaatatcgc tggacattac taataaaccc actataaaca catgtacata
     4981 tgtatgtttt ggcatacaat gagtagttgg ggaaaaaatg tgtaaaagca ccgtgaccat
     5041 cacagcataa agataaccag ctgaagtatc gaatatgagt aacccccaaa ttgaatcaca
     5101 tgccgcaact gataggaccc atggaagtac actcttcatg gcgatataca agacacacac
     5161 aagcacgaac acccagttgc ggaggaaatt ctccgtaaat gaaaacccaa tcggcgaaca
     5221 attcataccc atatatggta aaagttttga acgcgacttg agagcggaga gcattgcggc
     5281 tgataaggtt ttagcgctaa gcgggcttta taaaacgggc tgcgggacca gttttcatat
     5341 cactaccgtt tgagttcttg tgctgtgtgg atactcctcc cgacacaaag ccgctccatc
     5401 agccagcagt cgtctaatcc agagaccccg gatccagata tcgaggcctg tctagagaag
     5461 cttgttcgaa tctcgagtgc gcgcttccgg aggtatacac ctaggcggta ccactgcagt
     5521 gaattcggag ctccgccacc atggactaca aagaccatga cggtgattat aaagatcatg
     5581 acatcgatta caaggatgac gatgacaagc accggttgag ctccgccacc atggagcaaa
     5641 agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct gaagaggact
     5701 tgaatgaaat ggagcaaaag ctcatttctg aagaggactt gaatgaaatg gagcaaaagc
     5761 tcatttctga agaggacttg aatgaaatgg agcaaaagct catttctgaa gaggacttga
     5821 atgaaatgga gagcttgggc gacctcacca tggagcaaaa gctcatttct gaagaggact
     5881 tgaatcaccg gtatacaagt ttgtacaaaa aagcaggctc cgcggccgcc cccttcaccg
     5941 cacgcaagag ccacatgtac aacgcaatac ccgccggctt tgagtcggac gaggagaaca
     6001 tggagaacct aatgagcaac ctgaagatca agcgcttgga ggacatcacg acaggtgctg
     6061 gaatcgacgg ctgtaacttt gacgccacct tggacgcaaa ggcggaggag ttttttaagc
     6121 tgttccgcga gaagtggaac atgtatagca agaaaaagtc gccacatctg cgccaggaat
     6181 ttggaagggc tttaatgggc catcaggatc ctttgctcct ggccctgaag atcttcgcca
     6241 actgcccaga cagcagcaac atcaagacga agagcttgtc acattttgtg ctggacacgg
     6301 tttgcaagct ccacaaggat ttcccgcatc ttggcgaggg ctgtgatccc aacaccagca
     6361 tgatagcctt taactttgtc aagacctccg gcttgctggc cctcaacaat gctgttatcc
     6421 atgcctacag tctacgtcag atccgcgacc tactgcttcc caagttgcgc gaactcctgg
     6481 acaacgggct ttacaaagag gtcacccaat ggtccatcag tctgcagttg acgcacgagt
     6541 tcgacatgct ggaactggct ttcccgctga ttgcaattga gaaactacca ctggccgagg
     6601 aatatctgga ccatgccaca cagcagaggt tgccctttgt caagttcttg gattcgttgc
     6661 tgcacaagga gaagtcggtt ttagagctct gcgagcatct actcgatcgg tacaaaaatc
     6721 tcaaaatttc ccataacgtg ctcagctatc ggcctatggc caagattgtg gcgcgactgg
     6781 ccaagaagta tggttttgac gacgccgtca cgcccaacta taagttcacg aagacgtgca
     6841 gctacttgca ttacctttac cgcgagtacg agaagacgcg catgaacctg gccagtttcc
     6901 gggaggttgt gagcgtccat gcctttaacc acgagctgcg cacggacttc gtaaagtatt
     6961 tggcctctgc tggagctcat tctgaagcca tctactggta cacggagttc aatattgacc
     7021 cgaaagattg tccgttggag atcgagacgc aggtatcgca aaacggtgct ggaaaagcgt
     7081 caggctgaaa gggtgggcgc gccgacccag ctttc
//
