LOCUS       sequence-199078-        8796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FLAG Myc tagged A.thaliana-Hen1 (codon optimised for 
            Drosophila) in Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8796)
  AUTHORS   Alberti C, Manzenreither RA, Sowemimo I, Burkard TR, Wang J, 
            Mahofsky K, Ameres SL, Cochella L
  TITLE     Cell-type specific sequencing of microRNAs from complex animal 
            tissues.
  JOURNAL   Nat Methods. 2018 Feb 26. pii: nmeth.4610. doi: 10.1038/nmeth.4610.
  PUBMED    29481550
REFERENCE   2  (bases 1 to 8796)
  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"; journalName: "Nat 
            Methods. 2018 Feb 26. pii: nmeth.4610. doi: 10.1038/nmeth.4610."
COMMENT     Sequence Label: pAFMW-AthHEN1
FEATURES             Location/Qualifiers
     source          1..8796
                     /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..5427
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      5536..5545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5545..5610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      5626..5635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5635..5664
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5674..5703
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5713..5742
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5752..5781
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5791..5820
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5854..5883
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    5896..5920
                     /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(8786..8796,1..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 ctgtccttct
     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 aataaaacaa attatttgaa agagaattag
     4381 gaatcggacg cttatcgtta gggtaacaac aagaaatgct tactgagtca cagcctctgg
     4441 aaaactgccg caagccagag agagagagaa aaagagggag agcagcttag accgcatgtg
     4501 cttgtgtgtg aggcgtctct ctcttcgtct ctgttgcgca aacgcataga ctgcactgag
     4561 aaaatcgatt acctattttt tatgaatgaa tatttgcact attactattc aaaactatta
     4621 agatagcaat cacattcaat agccaaatac tataccacct gagcgatgca acgaaatgat
     4681 caatttgagc aaaaatgctg catatttagg acggcatcat tatagaaatg cttcttgctg
     4741 tgtacttttc tctcgtctgg cagctgtttc gccgttattg ttaaaaccgg cttaagttag
     4801 gtgtgttttc tacgactagt gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc
     4861 tggaataacc aatttgaagt gcagatagca gtaaacgtaa gctaatatga atattattta
     4921 actgtaatgt tttaatatcg ctggacatta ctaataaacc cactataaac acatgtacat
     4981 atgtatgttt tggcatacaa tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca
     5041 tcacagcata aagataacca gctgaagtat cgaatatgag taacccccaa attgaatcac
     5101 atgccgcaac tgataggacc catggaagta cactcttcat ggcgatatac aagacacaca
     5161 caagcacgaa cacccagttg cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac
     5221 aattcatacc catatatggt aaaagttttg aacgcgactt gagagcggag agcattgcgg
     5281 ctgataaggt tttagcgcta agcgggcttt ataaaacggg ctgcgggacc agttttcata
     5341 tcactaccgt ttgagttctt gtgctgtgtg gatactcctc ccgacacaaa gccgctccat
     5401 cagccagcag tcgtctaatc cagagacccc ggatccagat atcgaggcct gtctagagaa
     5461 gcttgttcga atctcgagtg cgcgcttccg gaggtataca cctaggcggt accactgcag
     5521 tgaattcgga gctccgccac catggactac aaagaccatg acggtgatta taaagatcat
     5581 gacatcgatt acaaggatga cgatgacaag caccggttga gctccgccac catggagcaa
     5641 aagctcattt ctgaagagga cttgaatgaa atggagcaaa agctcatttc tgaagaggac
     5701 ttgaatgaaa tggagcaaaa gctcatttct gaagaggact tgaatgaaat ggagcaaaag
     5761 ctcatttctg aagaggactt gaatgaaatg gagcaaaagc tcatttctga agaggacttg
     5821 aatgaaatgg agagcttggg cgacctcacc atggagcaaa agctcatttc tgaagaggac
     5881 ttgaatcacc ggtatacaag tttgtacaaa aaagcaggct ccgcggccgc ccccttcacc
     5941 atggcgggtg gtggcaagca caccccgact ccaaaggcta ttatccatca aaagtttggc
     6001 gctaaggcct cgtatacagt ggaggaagtt catgactcgt cgcagtcggg ctgcctgggc
     6061 ctggccattc cgcagaaggg tccgtgcctc taccggtgtc acctgcagct gccagaattc
     6121 tccgtggtga gcaatgtatt caaaaagaag aaggatagtg agcagagtgc tgctgagctg
     6181 gccttggata aactgggtat tcgaccccag aacgacgatt tgacagtgga tgaggcgcgc
     6241 gatgagatcg tgggcaggat caaatatata ttcagtgacg agttcctgag tgccgagcac
     6301 ccgttgggcg cacacttgcg cgcagccctg cgtcgcgatg gcgaacgttg cggctcggtt
     6361 ccggttagtg tgattgctac cgtggacgcc aaaattaaca gccgctgtaa gataatcaat
     6421 ccctccgtgg agagtgaccc gttcctggcc atttcgtacg tgatgaaagc agcagctaag
     6481 ctggccgact acatagtggc ctcgccacac ggcctccgtc gcaaaaacgc gtacccgtcc
     6541 gagatagtcg aggcgttggc gacgcacgta agcgattcgc tccattcccg ggaggtggcc
     6601 gctgtttaca ttccgtgcat cgatgaggag gttgtagagt tggatacgct ctacatcagc
     6661 tcgaatcgcc attacctgga ttcgatcgcc gagcgactcg gtttgaagga cggcaatcag
     6721 gttatgatta gtcgcatgtt tggcaaggcg tcgtgtggtt ccgagtgtcg tctgtactcg
     6781 gagatcccaa agaagtatct ggacaattcc agcgatgcca gcggtacttc caacgaggat
     6841 tccagccata tcgtcaagag ccgcaacgct cgcgcctcgt acatctgtgg tcaggatatc
     6901 catggtgacg ccatcctcgc cagcgtagga taccgatgga agtcggacga cctggactac
     6961 gatgatgtga cagtcaacag tttctacagg atatgctgcg gcatgtcccc gaatggtata
     7021 tacaaaataa gcaggcaggc cgtgatcgcg gcgcagctgc ccttcgcctt tactacgaag
     7081 agcaattggc gcggtccgtt gccccgtgag atcctgggtc tgttctgcca ccagcaccga
     7141 ctggcggaac caattctgtc cagcagtacc gcgccggtga agagtttgtc cgacatcttt
     7201 cgcagtcaca agaagttgaa ggtaagcggt gttgacgacg caaacgagaa cttgtcccgc
     7261 cagaaggaag atacgcccgg actgggtcac ggcttccgat gtgaggttaa gatttttacc
     7321 aagagtcagg acctggtact cgaatgctcc ccacgtaaat tctacgagaa ggagaacgat
     7381 gccatccaga acgcctcctt gaaggccttg ctctggttca gcaagttctt tgccgacctg
     7441 gacgttgacg gcgagcaaag ttgcgacact gatgatgatc aggatacgaa gagcagtagc
     7501 ccaaacgtat tcgccgcccc ccccatcttg cagaaggagc actcctcgga gagcaagaac
     7561 accaatgtgc tcagtgcgga aaagcgggtc cagtccatca ccaatggcag tgtcgtgagt
     7621 atttgctact ccctcagcct ggccgtcgat cccgagtatt cgtccgatgg cgaaagcccc
     7681 agggaggata atgaaagtaa cgaggagatg gaaagtgaat acagcgccaa ctgtgagtcg
     7741 agtgtggagc tgattgagtc gaacgaggag atcgagttcg aggtcggcac cggcagcatg
     7801 aacccgcaca ttgagagcga ggtgacccaa atgaccgttg gtgagtacgc cagctttcgc
     7861 atgacaccgc cagacgcggc agaggcactg atcctggcag tgggcagcga caccgtgcga
     7921 atccgctccc tgctgagcga gaggccatgc ctcaattaca acatcctgct gctgggcgtt
     7981 aagggaccca gtgaggagcg catggaggcc gccttcttca agccccctct ctcgaagcaa
     8041 cgagtggagt atgccctgaa gcatattcga gagtcgagtg catcgacgct ggtggatttt
     8101 ggctgtggct ccggaagtct gttggatagt ctcctggact acccaacaag cctgcaaacg
     8161 attataggcg tggacatctc gcctaagggc ctcgcccgtg ccgcgaagat gctgcacgtt
     8221 aaattgaaca aggaggcgtg caatgttaag agtgctactc tgtacgatgg aagcattctc
     8281 gagttcgaca gccgcctgca cgatgttgac attggcactt gtttggaagt gattgaacac
     8341 atggaagagg atcaagcctg cgagtttggt gaaaaggtgt tgagtttgtt ccacccgaag
     8401 ctgctcatag tgagcacccc caattatgaa tttaacacga tactgcagcg ctcgacgccc
     8461 gagacgcagg aggagaacaa ctccgagcca cagctcccga aattcaggaa ccatgaccat
     8521 aaattcgagt ggacacggga gcagtttaat caatgggctt cgaagctggg caagcgccac
     8581 aactactccg tggagttcag cggtgtcgga ggcagtggcg aggtggaacc gggctttgcc
     8641 agccagatcg ctatattccg gcgtgaagct agctcggtcg agaatgtcgc agaaagtagc
     8701 atgcaaccct acaaagtgat ttgggaatgg aagaaggagg acgtcgaaaa aaagaagact
     8761 gacttgtaaa agggtgggcg cgccgaccca gctttc
//
