LOCUS       sequence-375839-        7841 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 7841)
  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 7841)
  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
FEATURES             Location/Qualifiers
     source          1..7841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        140..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"
     regulatory      2847..2856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2856..2885
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2895..2924
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2934..2963
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2973..3002
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3012..3041
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3075..3104
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    3117..3141
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5053..5077)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5169..5234
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5364..5384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5809..5943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5846..5865)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5900..5919
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6040..6108,6109..6900)
                     /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(6258..6277)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7071..7659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7560..7579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7813..7830
                     /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 aaataaaaca aattatttga aagagaatta ggaatcggac gcttatcgtt
     1621 agggtaacaa caagaaatgc ttactgagtc acagcctctg gaaaactgcc gcaagccaga
     1681 gagagagaga aaaagaggga gagcagctta gaccgcatgt gcttgtgtgt gaggcgtctc
     1741 tctcttcgtc tctgttgcgc aaacgcatag actgcactga aaaaatcgat 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 gcaccggttg agctccgcca ccatggagca aaagctcatt tctgaagagg
     2881 acttgaatga aatggagcaa aagctcattt ctgaagagga cttgaatgaa atggagcaaa
     2941 agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct gaagaggact
     3001 tgaatgaaat ggagcaaaag ctcatttctg aagaggactt gaatgaaatg gagagcttgg
     3061 gcgacctcac catggagcaa aagctcattt ctgaagagga cttgaatcac cggtatacaa
     3121 gtttgtacaa aaaagcaggc tccgcggccg cccccttcac cgcacgcaag agccacatgt
     3181 acaacgcaat acccgccggc tttgagtcgg acgaggagaa catggagaac ctaatgagca
     3241 acctgaagat caagcgcttg gaggacatca cgacaggtgc tggaatcgac ggctgtaact
     3301 ttgacgccac cttggacgca aaggcggagg agttttttaa gctgttccgc gagaagtgga
     3361 acatgtatag caagaaaaag tcgccacatc tgcgccagga atttggaagg gctttaatgg
     3421 gccatcagga tcctttgctc ctggccctga agatcttcgc caactgccca gacagcagca
     3481 acatcaagac gaagagcttg tcacattttg tgctggacac ggtttgcaag ctccacaagg
     3541 atttcccgca tcttggcgag ggctgtgatc ccaacaccag catgatagcc tttaactttg
     3601 tcaagacctc cggcttgctg gccctcaaca atgctgttat ccatgcctac agtctacgtc
     3661 agatccgcga cctactgctt cccaagttgc gcgaactcct ggacaacggg ctttacaaag
     3721 aggtcaccca atggtccatc agtctgcagt tgacgcacga gttcgacatg ctggaactgg
     3781 ctttcccgct gattgcaatt gagaaactac cactggccga ggaatatctg gaccatgcca
     3841 cacagcagag gttgcccttt gtcaagttct tggattcgtt gctgcacaag gagaagtcgg
     3901 ttttagagct ctgcgagcat ctactcgatc ggtacaaaaa tctcaaaatt tcccataacg
     3961 tgctcagcta tcggcctatg gccaagattg tggcgcgact ggccaagaag tatggttttg
     4021 acgacgccgt cacgcccaac tataagttca cgaagacgtg cagctacttg cattaccttt
     4081 accgcgagta cgagaagacg cgcatgaacc tggccagttt ccgggaggtt gtgagcgtcc
     4141 atgcctttaa ccacgagctg cgcacggact tcgtaaagta tttggcctct gctggagctc
     4201 attctgaagc catctactgg tacacggagt tcaatattga cccgaaagat tgtccgttgg
     4261 agatcgagac gcaggtatcg caaaacggtg ctggaaaagc gtcaggctgg gaatcccccg
     4321 gcaaggaacg ctgtccttcg agtagatgtg atatgtacct gaccatggac ctgcccgacg
     4381 agtgccttat cattgtgaac aaggctgatg agttcgatcg tatgttatac catctgcagc
     4441 aggagtgcgt catatacctg gattctgaat ggatgcagag cgtgtgcgga gacaatcagc
     4501 tgtgcgtgct acagatcgcc accggccaca atgtctacct gatcgattgc ctggcgcggg
     4561 agagcttacg ctcggaacac tggcgcttgc tgggcgcgaa tatctttaat aacgtgaaca
     4621 tccgtaaggt ggggttctct atggtcagtg atctcagtgt attgcagcgg tcactgcccc
     4681 tgcaactgcg ccttcaaatg cctcaccact acttggacct tcgcaatctc tggttggagt
     4741 taaaaaagca gcgcttcggc gttgaacttc ccttcggcaa tgttaaccgg gcaggagacg
     4801 cccttacgga tctctcattg gcatgccttg gcaagaaact gaacaaatca aaccagtgct
     4861 caaactgggc caatagaccg ctgcgtcgcg agcagattct ttatgcagcc atcgatgcgc
     4921 ggtgtttgat gctgatctac aacacgctca ttgaacgcgt gtccttcata caagcggtta
     4981 tcgaaaagag catcgccagc aataactttc tcaggcgggg tgcccatgtt aagtgaaagg
     5041 gtgggcgcgc cgacccagct ttcttgtaca aagtggtgac gtaagctagc aggatctttg
     5101 tgaaggaacc ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa
     5161 gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg
     5221 tttgtgtatt ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct
     5281 ttaatgagga aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg
     5341 ctgactctca acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact
     5401 ttccttcaga attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt
     5461 gctttgctat ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa
     5521 aatattctgt aacctttata agtaggcata acagttataa tcataacata ctgttttttc
     5581 ttactccaca caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct
     5641 ttagcttttt aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta
     5701 gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     5761 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     5821 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5881 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     5941 atcccgttta aactacgcgt aattcaaaca gggttctggc gtcgttctcg tactgttttc
     6001 cccaggccag tgctttagcg ttattgaaaa aggaagagta tgagtattca acatttccgt
     6061 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6121 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6181 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6241 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6301 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6361 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6421 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6481 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6541 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6601 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6661 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6721 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6781 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6841 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6901 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6961 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7021 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7081 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7141 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7201 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7261 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7321 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7381 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7441 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7501 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7561 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7621 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7681 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7741 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7801 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag a
//
