LOCUS       sequence-375861-        7842 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 7842)
  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 7842)
  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[R272A,K276A]
FEATURES             Location/Qualifiers
     source          1..7842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        140..2649
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      2758..2767
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2767..2832
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2848..2857
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2857..2886
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2896..2925
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2935..2964
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2974..3003
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3013..3042
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3076..3105
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    3118..3142
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5054..5078)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5170..5235
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5365..5385
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5810..5944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5847..5866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5901..5920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6041..6109,6110..6901)
                     /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(6259..6278)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7072..7660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7561..7580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7814..7831
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaggcaact
       61 cgtgaaaggt aggcggatca gcggccgcag catgcaattc tatattctaa aaacacaaat
      121 gatacttcta aaaaaaaatc atgaatggca tcaactctga atcaaatctt tgcagatgca
      181 cctacttctc atttccactg tcacatcatt tttccagatc tcgctgcctg ttatgtggcc
      241 cacaaaccaa gacacgtttt atggccatta aagctggctg atcgtcgcca aacaccaaat
      301 acataatgaa tatgtacaca ttcgagaaag aagcgatcaa agaagcgtct tcgggcggag
      361 taggagaatg cggaggagaa ggagaacgag ctgatctagt atctctccac aatccaatgc
      421 caactgacca actggccata ttcggagcaa tttgaagcca atttccatcg cctggcgatc
      481 gctccattct tggctatatg tttttcaccg ttacccgggg ccattttcaa agactcgtcg
      541 gcaagataag attgtgtcac tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt
      601 tcgcgatgac gtcggcgagt attttgaaga atgagaataa tttgtattta tacgaaaatc
      661 agttagtgga attttctaca aaaacatgtt atctatagat aattttgttg caaaatatgt
      721 tgactatgac aaagattgta tgtatatacc tttaatgtat tctcattttc ttatgtattt
      781 ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa aggcttgaat
      841 ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga atattcaatt
      901 ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa gtaaagccaa
      961 aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg
     1021 cagcggaagg aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg
     1081 acgttggagc cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc
     1141 gccggttgtt tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc
     1201 tcaacgcacc gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt
     1261 atttaaacaa agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact
     1321 tattgccatg tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc
     1381 tctttcactt tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat
     1441 tacgctgaca aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg
     1501 catttgcaat aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca
     1561 cacaaaaaaa aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt
     1621 tagggtaaca acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag
     1681 agagagagag aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct
     1741 ctctcttcgt ctctgttgcg caaacgcata gactgcactg aaaaaatcga ttacctattt
     1801 tttatgaatg aatatttgca ctattactat tcaaaactat taagatagca atcacattca
     1861 atagccaaat actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc
     1921 tgcatattta ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct
     1981 ggcagctgtt tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta
     2041 gtgaatgccc tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa
     2101 gtgcagatag cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat
     2161 cgctggacat tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac
     2221 aatgagtagt tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac
     2281 cagctgaagt atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga
     2341 cccatggaag tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt
     2401 tgcggaggaa attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg
     2461 gtaaaagttt tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc
     2521 taagcgggct ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc
     2581 ttgtgctgtg tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa
     2641 tccagagacc ccggatccag atatcgaggc ctgtctagag aagcttgttc gaatctcgag
     2701 tgcgcgcttc cggaggtata cacctaggcg gtaccactgc agtgaattcg gagctccgcc
     2761 accatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
     2821 gacgatgaca agcaccggtt gagctccgcc accatggagc aaaagctcat ttctgaagag
     2881 gacttgaatg aaatggagca aaagctcatt tctgaagagg acttgaatga aatggagcaa
     2941 aagctcattt ctgaagagga cttgaatgaa atggagcaaa agctcatttc tgaagaggac
     3001 ttgaatgaaa tggagcaaaa gctcatttct gaagaggact tgaatgaaat ggagagcttg
     3061 ggcgacctca ccatggagca aaagctcatt tctgaagagg acttgaatca ccggtataca
     3121 agtttgtaca aaaaagcagg ctccgcggcc gcccccttca ccgcacgcaa gagccacatg
     3181 tacaacgcaa tacccgccgg ctttgagtcg gacgaggaga acatggagaa cctaatgagc
     3241 aacctgaaga tcaagcgctt ggaggacatc acgacaggtg ctggaatcga cggctgtaac
     3301 tttgacgcca ccttggacgc aaaggcggag gagtttttta agctgttccg cgagaagtgg
     3361 aacatgtata gcaagaaaaa gtcgccacat ctgcgccagg aatttggaag ggctttaatg
     3421 ggccatcagg atcctttgct cctggccctg aagatcttcg ccaactgccc agacagcagc
     3481 aacatcaaga cgaagagctt gtcacatttt gtgctggaca cggtttgcaa gctccacaag
     3541 gatttcccgc atcttggcga gggctgtgat cccaacacca gcatgatagc ctttaacttt
     3601 gtcaagacct ccggcttgct ggccctcaac aatgctgtta tccatgccta cagtctacgt
     3661 cagatccgcg acctactgct tcccaagttg cgcgaactcc tggacaacgg gctttacaaa
     3721 gaggtcaccc aatggtccat cagtctgcag ttgacgcacg agttcgacat gctggaactg
     3781 gctttcccgc tgattgcaat tgagaaacta ccactggccg aggaatatct ggaccatgcc
     3841 acacagcaga ggttgccctt tgtcaagttc ttggattcgt tgctgcacaa ggagaagtcg
     3901 gttttagagc tctgcgagca tctactcgat cggtacaaaa atctcaaaat ttcccataac
     3961 gtgctcagct atgcgcctat ggccgcgatt gtggcgcgac tggccaagaa gtatggtttt
     4021 gacgacgccg tcacgcccaa ctataagttc acgaagacgt gcagctactt gcattacctt
     4081 taccgcgagt acgagaagac gcgcatgaac ctggccagtt tccgggaggt tgtgagcgtc
     4141 catgccttta accacgagct gcgcacggac ttcgtaaagt atttggcctc tgctggagct
     4201 cattctgaag ccatctactg gtacacggag ttcaatattg acccgaaaga ttgtccgttg
     4261 gagatcgaga cgcaggtatc gcaaaacggt gctggaaaag cgtcaggctg ggaatccccc
     4321 ggcaaggaac gctgtccttc gagtagatgt gatatgtacc tgaccatgga cctgcccgac
     4381 gagtgcctta tcattgtgaa caaggctgat gagttcgatc gtatgttata ccatctgcag
     4441 caggagtgcg tcatatacct ggattctgaa tggatgcaga gcgtgtgcgg agacaatcag
     4501 ctgtgcgtgc tacagatcgc caccggccac aatgtctacc tgatcgattg cctggcgcgg
     4561 gagagcttac gctcggaaca ctggcgcttg ctgggcgcga atatctttaa taacgtgaac
     4621 atccgtaagg tggggttctc tatggtcagt gatctcagtg tattgcagcg gtcactgccc
     4681 ctgcaactgc gccttcaaat gcctcaccac tacttggacc ttcgcaatct ctggttggag
     4741 ttaaaaaagc agcgcttcgg cgttgaactt cccttcggca atgttaaccg ggcaggagac
     4801 gcccttacgg atctctcatt ggcatgcctt ggcaagaaac tgaacaaatc aaaccagtgc
     4861 tcaaactggg ccaatagacc gctgcgtcgc gagcagattc tttatgcagc catcgatgcg
     4921 cggtgtttga tgctgatcta caacacgctc attgaacgcg tgtccttcat acaagcggtt
     4981 atcgaaaaga gcatcgccag caataacttt ctcaggcggg gtgcccatgt taagtgaaag
     5041 ggtgggcgcg ccgacccagc tttcttgtac aaagtggtga cgtaagctag caggatcttt
     5101 gtgaaggaac cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa
     5161 agctctaagg taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt
     5221 gtttgtgtat tttagattcc aacctatgga actgatgaat gggagcagtg gtggaatgcc
     5281 tttaatgagg aaaacctgtt ttgctcagaa gaaatgccat ctagtgatga tgaggctact
     5341 gctgactctc aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac
     5401 tttccttcag aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct
     5461 tgctttgcta tttacaccac aaaggaaaaa gctgcactgc tatacaagaa aattatggaa
     5521 aaatattctg taacctttat aagtaggcat aacagttata atcataacat actgtttttt
     5581 cttactccac acaggcatag agtgtctgct attaataact atgctcaaaa attgtgtacc
     5641 tttagctttt taatttgtaa aggggttaat aaggaatatt tgatgtatag tgccttgact
     5701 agagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     5761 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     5821 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5881 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5941 gatcccgttt aaactacgcg taattcaaac agggttctgg cgtcgttctc gtactgtttt
     6001 ccccaggcca gtgctttagc gttattgaaa aaggaagagt atgagtattc aacatttccg
     6061 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6121 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6181 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6241 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6301 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6361 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6421 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6481 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6541 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6601 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6661 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6721 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6781 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6841 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6901 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6961 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7021 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7081 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7141 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7201 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7261 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7321 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7381 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7441 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7501 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7561 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7621 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7681 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7741 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7801 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa ga
//
