LOCUS       sequence-375856-        7840 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 7840)
  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 7840)
  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[K284A]
FEATURES             Location/Qualifiers
     source          1..7840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        140..2647
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      2756..2765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2765..2830
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2846..2855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2855..2884
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2894..2923
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2933..2962
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2972..3001
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3011..3040
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3074..3103
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    3116..3140
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5052..5076)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5168..5233
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5363..5383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5808..5942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5845..5864)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5899..5918
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6039..6107,6108..6899)
                     /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(6257..6276)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7070..7658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7559..7578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7812..7829
                     /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 agagagagaa aaagagggag agcagcttag accgcatgtg cttgtgtgtg aggcgtctct
     1741 ctcttcgtct ctgttgcgca aacgcataga ctgcactgaa aaaatcgatt acctattttt
     1801 tatgaatgaa tatttgcact attactattc aaaactatta agatagcaat cacattcaat
     1861 agccaaatac tataccacct gagcgatgca acgaaatgat caatttgagc aaaaatgctg
     1921 catatttagg acggcatcat tatagaaatg cttcttgctg tgtacttttc tctcgtctgg
     1981 cagctgtttc gccgttattg ttaaaaccgg cttaagttag gtgtgttttc tacgactagt
     2041 gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc tggaataacc aatttgaagt
     2101 gcagatagca gtaaacgtaa gctaatatga atattattta actgtaatgt tttaatatcg
     2161 ctggacatta ctaataaacc cactataaac acatgtacat atgtatgttt tggcatacaa
     2221 tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca tcacagcata aagataacca
     2281 gctgaagtat cgaatatgag taacccccaa attgaatcac atgccgcaac tgataggacc
     2341 catggaagta cactcttcat ggcgatatac aagacacaca caagcacgaa cacccagttg
     2401 cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac aattcatacc catatatggt
     2461 aaaagttttg aacgcgactt gagagcggag agcattgcgg ctgataaggt tttagcgcta
     2521 agcgggcttt ataaaacggg ctgcgggacc agttttcata tcactaccgt ttgagttctt
     2581 gtgctgtgtg gatactcctc ccgacacaaa gccgctccat cagccagcag tcgtctaatc
     2641 cagagacccc ggatccagat atcgaggcct gtctagagaa gcttgttcga atctcgagtg
     2701 cgcgcttccg gaggtataca cctaggcggt accactgcag tgaattcgga gctccgccac
     2761 catggactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga
     2821 cgatgacaag caccggttga gctccgccac catggagcaa aagctcattt ctgaagagga
     2881 cttgaatgaa atggagcaaa agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa
     2941 gctcatttct gaagaggact tgaatgaaat ggagcaaaag ctcatttctg aagaggactt
     3001 gaatgaaatg gagcaaaagc tcatttctga agaggacttg aatgaaatgg agagcttggg
     3061 cgacctcacc atggagcaaa agctcatttc tgaagaggac ttgaatcacc ggtatacaag
     3121 tttgtacaaa aaagcaggct ccgcggccgc ccccttcacc gcacgcaaga gccacatgta
     3181 caacgcaata cccgccggct ttgagtcgga cgaggagaac atggagaacc taatgagcaa
     3241 cctgaagatc aagcgcttgg aggacatcac gacaggtgct ggaatcgacg gctgtaactt
     3301 tgacgccacc ttggacgcaa aggcggagga gttttttaag ctgttccgcg agaagtggaa
     3361 catgtatagc aagaaaaagt cgccacatct gcgccaggaa tttggaaggg ctttaatggg
     3421 ccatcaggat cctttgctcc tggccctgaa gatcttcgcc aactgcccag acagcagcaa
     3481 catcaagacg aagagcttgt cacattttgt gctggacacg gtttgcaagc tccacaagga
     3541 tttcccgcat cttggcgagg gctgtgatcc caacaccagc atgatagcct ttaactttgt
     3601 caagacctcc ggcttgctgg ccctcaacaa tgctgttatc catgcctaca gtctacgtca
     3661 gatccgcgac ctactgcttc ccaagttgcg cgaactcctg gacaacgggc tttacaaaga
     3721 ggtcacccaa tggtccatca gtctgcagtt gacgcacgag ttcgacatgc tggaactggc
     3781 tttcccgctg attgcaattg agaaactacc actggccgag gaatatctgg accatgccac
     3841 acagcagagg ttgccctttg tcaagttctt ggattcgttg ctgcacaagg agaagtcggt
     3901 tttagagctc tgcgagcatc tactcgatcg gtacaaaaat ctcaaaattt cccataacgt
     3961 gctcagctat cggcctatgg ccaagattgt ggcgcgactg gccaaggcgt atggttttga
     4021 cgacgccgtc acgcccaact ataagttcac gaagacgtgc agctacttgc attaccttta
     4081 ccgcgagtac gagaagacgc gcatgaacct ggccagtttc cgggaggttg tgagcgtcca
     4141 tgcctttaac cacgagctgc gcacggactt cgtaaagtat ttggcctctg ctggagctca
     4201 ttctgaagcc atctactggt acacggagtt caatattgac ccgaaagatt gtccgttgga
     4261 gatcgagacg caggtatcgc aaaacggtgc tggaaaagcg tcaggctggg aatcccccgg
     4321 caaggaacgc tgtccttcga gtagatgtga tatgtacctg accatggacc tgcccgacga
     4381 gtgccttatc attgtgaaca aggctgatga gttcgatcgt atgttatacc atctgcagca
     4441 ggagtgcgtc atatacctgg attctgaatg gatgcagagc gtgtgcggag acaatcagct
     4501 gtgcgtgcta cagatcgcca ccggccacaa tgtctacctg atcgattgcc tggcgcggga
     4561 gagcttacgc tcggaacact ggcgcttgct gggcgcgaat atctttaata acgtgaacat
     4621 ccgtaaggtg gggttctcta tggtcagtga tctcagtgta ttgcagcggt cactgcccct
     4681 gcaactgcgc cttcaaatgc ctcaccacta cttggacctt cgcaatctct ggttggagtt
     4741 aaaaaagcag cgcttcggcg ttgaacttcc cttcggcaat gttaaccggg caggagacgc
     4801 ccttacggat ctctcattgg catgccttgg caagaaactg aacaaatcaa accagtgctc
     4861 aaactgggcc aatagaccgc tgcgtcgcga gcagattctt tatgcagcca tcgatgcgcg
     4921 gtgtttgatg ctgatctaca acacgctcat tgaacgcgtg tccttcatac aagcggttat
     4981 cgaaaagagc atcgccagca ataactttct caggcggggt gcccatgtta agtgaaaggg
     5041 tgggcgcgcc gacccagctt tcttgtacaa agtggtgacg taagctagca ggatctttgt
     5101 gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag agatttaaag
     5161 ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt
     5221 ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt ggaatgcctt
     5281 taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg aggctactgc
     5341 tgactctcaa cattctactc ctccaaaaaa gaagagaaag gtagaagacc ccaaggactt
     5401 tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg
     5461 ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa
     5521 atattctgta acctttataa gtaggcataa cagttataat cataacatac tgttttttct
     5581 tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat tgtgtacctt
     5641 tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg ccttgactag
     5701 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5761 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5821 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5881 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     5941 tcccgtttaa actacgcgta attcaaacag ggttctggcg tcgttctcgt actgttttcc
     6001 ccaggccagt gctttagcgt tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6061 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6121 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6181 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6241 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6301 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6361 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6421 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6481 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6541 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6601 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6661 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6721 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6781 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6841 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6901 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6961 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7021 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7081 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7141 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7201 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7261 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7321 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7381 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7441 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7501 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7561 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7621 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7681 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7741 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7801 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
