LOCUS       sequence-375982-        7845 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 7845)
  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 7845)
  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, R280A,K284A]
FEATURES             Location/Qualifiers
     source          1..7845
                     /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(5057..5081)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5173..5238
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5368..5388
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5813..5947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5850..5869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5904..5923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6044..6112,6113..6904)
                     /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(6262..6281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7075..7663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7564..7583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7817..7834
                     /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 ccatggcacg caagagccac
     3181 atgtacaacg caatacccgc cggctttgag tcggacgagg agaacatgga gaacctaatg
     3241 agcaacctga agatcaagcg cttggaggac atcacgacag gtgctggaat cgacggctgt
     3301 aactttgacg ccaccttgga cgcaaaggcg gaggagtttt ttaagctgtt ccgcgagaag
     3361 tggaacatgt atagcaagaa aaagtcgcca catctgcgcc aggaatttgg aagggcttta
     3421 atgggccatc aggatccttt gctcctggcc ctgaagatct tcgccaactg cccagacagc
     3481 agcaacatca agacgaagag cttgtcacat tttgtgctgg acacggtttg caagctccac
     3541 aaggatttcc cgcatcttgg cgagggctgt gatcccaaca ccagcatgat agcctttaac
     3601 tttgtcaaga cctccggctt gctggccctc aacaatgctg ttatccatgc ctacagtcta
     3661 cgtcagatcc gcgacctact gcttcccaag ttgcgcgaac tcctggacaa cgggctttac
     3721 aaagaggtca cccaatggtc catcagtctg cagttgacgc acgagttcga catgctggaa
     3781 ctggctttcc cgctgattgc aattgagaaa ctaccactgg ccgaggaata tctggaccat
     3841 gccacacagc agaggttgcc ctttgtcaag ttcttggatt cgttgctgca caaggagaag
     3901 tcggttttag agctctgcga gcatctactc gatcggtaca aaaatctcaa aatttcccat
     3961 aacgtgctca gctatgcgcc tatggccgcg attgtggcgg cactggccaa ggcgtatggt
     4021 tttgacgacg ccgtcacgcc caactataag ttcacgaaga cgtgcagcta cttgcattac
     4081 ctttaccgcg agtacgagaa gacgcgcatg aacctggcca gtttccggga ggttgtgagc
     4141 gtccatgcct ttaaccacga gctgcgcacg gacttcgtaa agtatttggc ctctgctgga
     4201 gctcattctg aagccatcta ctggtacacg gagttcaata ttgacccgaa agattgtccg
     4261 ttggagatcg agacgcaggt atcgcaaaac ggtgctggaa aagcgtcagg ctgggaatcc
     4321 cccggcaagg aacgctgtcc ttcgagtaga tgtgatatgt acctgaccat ggacctgccc
     4381 gacgagtgcc ttatcattgt gaacaaggct gatgagttcg atcgtatgtt ataccatctg
     4441 cagcaggagt gcgtcatata cctggattct gaatggatgc agagcgtgtg cggagacaat
     4501 cagctgtgcg tgctacagat cgccaccggc cacaatgtct acctgatcga ttgcctggcg
     4561 cgggagagct tacgctcgga acactggcgc ttgctgggcg cgaatatctt taataacgtg
     4621 aacatccgta aggtggggtt ctctatggtc agtgatctca gtgtattgca gcggtcactg
     4681 cccctgcaac tgcgccttca aatgcctcac cactacttgg accttcgcaa tctctggttg
     4741 gagttaaaaa agcagcgctt cggcgttgaa cttcccttcg gcaatgttaa ccgggcagga
     4801 gacgccctta cggatctctc attggcatgc cttggcaaga aactgaacaa atcaaaccag
     4861 tgctcaaact gggccaatag accgctgcgt cgcgagcaga ttctttatgc agccatcgat
     4921 gcgcggtgtt tgatgctgat ctacaacacg ctcattgaac gcgtgtcctt catacaagcg
     4981 gttatcgaaa agagcatcgc cagcaataac tttctcaggc ggggtgccca tgttaagtga
     5041 aagggtgggc gcgccgaccc agctttcttg tacaaagtgg tgacgtaagc tagcaggatc
     5101 tttgtgaagg aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt
     5161 taaagctcta aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta
     5221 attgtttgtg tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat
     5281 gcctttaatg aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct
     5341 actgctgact ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag
     5401 gactttcctt cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt
     5461 gcttgctttg ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg
     5521 gaaaaatatt ctgtaacctt tataagtagg cataacagtt ataatcataa catactgttt
     5581 tttcttactc cacacaggca tagagtgtct gctattaata actatgctca aaaattgtgt
     5641 acctttagct ttttaatttg taaaggggtt aataaggaat atttgatgta tagtgccttg
     5701 actagagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     5761 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     5821 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5881 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5941 ctggatcccg tttaaactac gcgtaattca aacagggttc tggcgtcgtt ctcgtactgt
     6001 tttccccagg ccagtgcttt agcgttattg aaaaaggaag agtatgagta ttcaacattt
     6061 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6121 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6181 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6241 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6301 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6361 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6421 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6481 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6541 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6601 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6661 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6721 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6781 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6841 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6901 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7201 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7681 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7741 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7801 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaga
//
