LOCUS       sequence-202483-        8796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FLAG Myc tagged catalytic dead 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-CM
FEATURES             Location/Qualifiers
     source          1..8796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          105..170
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             300..320
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    745..879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(782..801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     836..855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(976..1044,1045..1836)
                     /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(1194..1213)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2007..2595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2496..2515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2749..2766
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2917..5426
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     regulatory      5535..5544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5544..5609
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      5625..5634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5634..5663
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5673..5702
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5712..5741
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5751..5780
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5790..5819
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5853..5882
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    5895..5919
                     /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(8785..8796,1..13))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tgtacaaagt ggtgacgtaa gctagcagga tctttgtgaa ggaaccttac ttctgtggtg
       61 tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt
      121 taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct
      181 atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct
      241 cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc
      301 caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt
      361 tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg
      421 aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta
      481 ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt
      541 ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg
      601 ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca
      661 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
      721 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
      781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
      841 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc cgtttaaact acgcgtaatt
      901 caaacagggt tctggcgtcg ttctcgtact gttttcccca ggccagtgct ttagcgttat
      961 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1021 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1081 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1141 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1201 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1261 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1321 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1381 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1441 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1501 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1561 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1621 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     1681 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     1741 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     1801 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1861 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1921 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1981 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2041 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2101 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2161 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2221 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2281 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2341 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2401 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2461 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2521 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2581 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     2641 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2701 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     2761 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     2821 cattaatgca ggcaactcgt gaaaggtagg cggatcagcg gccgcagcat gcaattctat
     2881 attctaaaaa cacaaatgat acttctaaaa aaaaatcatg aatggcatca actctgaatc
     2941 aaatctttgc agatgcacct acttctcatt tccactgtca catcattttt ccagatctcg
     3001 ctgcctgtta tgtggcccac aaaccaagac acgttttatg gccattaaag ctggctgatc
     3061 gtcgccaaac accaaataca taatgaatat gtacacattc gagaaagaag cgatcaaaga
     3121 agcgtcttcg ggcggagtag gagaatgcgg aggagaagga gaacgagctg atctagtatc
     3181 tctccacaat ccaatgccaa ctgaccaact ggccatattc ggagcaattt gaagccaatt
     3241 tccatcgcct ggcgatcgct ccattcttgg ctatatgttt ttcaccgtta cccggggcca
     3301 ttttcaaaga ctcgtcggca agataagatt gtgtcactcg ctgtctctct tcatttgtcg
     3361 aagaatgctg aggaatttcg cgatgacgtc ggcgagtatt ttgaagaatg agaataattt
     3421 gtatttatac gaaaatcagt tagtggaatt ttctacaaaa acatgttatc tatagataat
     3481 tttgttgcaa aatatgttga ctatgacaaa gattgtatgt atataccttt aatgtattct
     3541 cattttctta tgtatttata atggcaatga tgatactgat gatattttaa gatgatgcca
     3601 gaccaaaagg cttgaatttc tgcgtctttt gccgaacgca gtgcatgtgc aattgttgtt
     3661 ttttggaata ttcaattttc ggactgtccg ctttgatttc agtttcttgg cttattcaaa
     3721 aagcaaagta aagccaaaaa agcgagatgg caataccaaa tgcggcaaaa cggtagtgga
     3781 aggaaagggg tgcggggcag cggaaggaag ggtggggcgg ggcgtggcgg ggtctgtggc
     3841 tgggcgcgac gtcaccgacg ttggagccac tcctttgacc atgtgtgcgt gtgtgtatta
     3901 ttcgtgtctc gccactcgcc ggttgttttt ttctttttat gctgcgctct ctctagcgcc
     3961 atctcgctta cgcatgctca acgcaccgca tgttgccgtt tccttttatg cgtcattttg
     4021 gctcgaaata ggcaattatt taaacaaaga ttagtcaacg aaaacgctaa aataaataag
     4081 tctacaatat ggttacttat tgccatgtgt gtgcagccaa cgatagcaac aaaagcaaca
     4141 acacaggtgg ctttccctct ttcacttttt gtttgcaagc cgcgtgcgag caagacggca
     4201 cgaccggcaa acgcaattac gctgacaaag agcagacgaa gttttggcga aaaacatcaa
     4261 ggcgcctgat acgaatgcat ttgcaataac aattgcgata tttaatattg tttatgaagc
     4321 tgtttgactt caaaacacac aaaaaaaaaa ataaaacaaa ttatttgaaa gagaattagg
     4381 aatcggacgc ttatcgttag ggtaacaaca agaaatgctt actgagtcac agcctctgga
     4441 aaactgccgc aagccagaga gagagagaaa aagagggaga gcagcttaga ccgcatgtgc
     4501 ttgtgtgtga ggcgtctctc tcttcgtctc tgttgcgcaa acgcatagac tgcactgaaa
     4561 aaatcgatta cctatttttt atgaatgaat atttgcacta ttactattca aaactattaa
     4621 gatagcaatc acattcaata gccaaatact ataccacctg agcgatgcaa cgaaatgatc
     4681 aatttgagca aaaatgctgc atatttagga cggcatcatt atagaaatgc ttcttgctgt
     4741 gtacttttct ctcgtctggc agctgtttcg ccgttattgt taaaaccggc ttaagttagg
     4801 tgtgttttct acgactagtg aatgccctac tagaagatgt gtgttgcaca aaatgtccct
     4861 ggaataacca atttgaagtg cagatagcag taaacgtaag ctaatatgaa tattatttaa
     4921 ctgtaatgtt ttaatatcgc tggacattac taataaaccc actataaaca catgtacata
     4981 tgtatgtttt ggcatacaat gagtagttgg ggaaaaaatg tgtaaaagca ccgtgaccat
     5041 cacagcataa agataaccag ctgaagtatc gaatatgagt aacccccaaa ttgaatcaca
     5101 tgccgcaact gataggaccc atggaagtac actcttcatg gcgatataca agacacacac
     5161 aagcacgaac acccagttgc ggaggaaatt ctccgtaaat gaaaacccaa tcggcgaaca
     5221 attcataccc atatatggta aaagttttga acgcgacttg agagcggaga gcattgcggc
     5281 tgataaggtt ttagcgctaa gcgggcttta taaaacgggc tgcgggacca gttttcatat
     5341 cactaccgtt tgagttcttg tgctgtgtgg atactcctcc cgacacaaag ccgctccatc
     5401 agccagcagt cgtctaatcc agagaccccg gatccagata tcgaggcctg tctagagaag
     5461 cttgttcgaa tctcgagtgc gcgcttccgg aggtatacac ctaggcggta ccactgcagt
     5521 gaattcggag ctccgccacc atggactaca aagaccatga cggtgattat aaagatcatg
     5581 acatcgatta caaggatgac gatgacaagc accggttgag ctccgccacc atggagcaaa
     5641 agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct gaagaggact
     5701 tgaatgaaat ggagcaaaag ctcatttctg aagaggactt gaatgaaatg gagcaaaagc
     5761 tcatttctga agaggacttg aatgaaatgg agcaaaagct catttctgaa gaggacttga
     5821 atgaaatgga gagcttgggc gacctcacca tggagcaaaa gctcatttct gaagaggact
     5881 tgaatcaccg gtatacaagt ttgtacaaaa aagcaggctc cgcggccgcc cccttcacca
     5941 tggcgggtgg tggcaagcac accccgactc caaaggctat tatccatcaa aagtttggcg
     6001 ctaaggcctc gtatacagtg gaggaagttc atgactcgtc gcagtcgggc tgcctgggcc
     6061 tggccattcc gcagaagggt ccgtgcctct accggtgtca cctgcagctg ccagaattct
     6121 ccgtggtgag caatgtattc aaaaagaaga aggatagtga gcagagtgct gctgagctgg
     6181 ccttggataa actgggtatt cgaccccaga acgacgattt gacagtggat gaggcgcgcg
     6241 atgagatcgt gggcaggatc aaatatatat tcagtgacga gttcctgagt gccgagcacc
     6301 cgttgggcgc acacttgcgc gcagccctgc gtcgcgatgg cgaacgttgc ggctcggttc
     6361 cggttagtgt gattgctacc gtggacgcca aaattaacag ccgctgtaag ataatcaatc
     6421 cctccgtgga gagtgacccg ttcctggcca tttcgtacgt gatgaaagca gcagctaagc
     6481 tggccgacta catagtggcc tcgccacacg gcctccgtcg caaaaacgcg tacccgtccg
     6541 agatagtcga ggcgttggcg acgcacgtaa gcgattcgct ccattcccgg gaggtggccg
     6601 ctgtttacat tccgtgcatc gatgaggagg ttgtagagtt ggatacgctc tacatcagct
     6661 cgaatcgcca ttacctggat tcgatcgccg agcgactcgg tttgaaggac ggcaatcagg
     6721 ttatgattag tcgcatgttt ggcaaggcgt cgtgtggttc cgagtgtcgt ctgtactcgg
     6781 agatcccaaa gaagtatctg gacaattcca gcgatgccag cggtacttcc aacgaggatt
     6841 ccagccatat cgtcaagagc cgcaacgctc gcgcctcgta catctgtggt caggatatcc
     6901 atggtgacgc catcctcgcc agcgtaggat accgatggaa gtcggacgac ctggactacg
     6961 atgatgtgac agtcaacagt ttctacagga tatgctgcgg catgtccccg aatggtatat
     7021 acaaaataag caggcaggcc gtgatcgcgg cgcagctgcc cttcgccttt actacgaaga
     7081 gcaattggcg cggtccgttg ccccgtgaga tcctgggtct gttctgccac cagcaccgac
     7141 tggcggaacc aattctgtcc agcagtaccg cgccggtgaa gagtttgtcc gacatctttc
     7201 gcagtcacaa gaagttgaag gtaagcggtg ttgacgacgc aaacgagaac ttgtcccgcc
     7261 agaaggaaga tacgcccgga ctgggtcacg gcttccgatg tgaggttaag atttttacca
     7321 agagtcagga cctggtactc gaatgctccc cacgtaaatt ctacgagaag gagaacgatg
     7381 ccatccagaa cgcctccttg aaggccttgc tctggttcag caagttcttt gccgacctgg
     7441 acgttgacgg cgagcaaagt tgcgacactg atgatgatca ggatacgaag agcagtagcc
     7501 caaacgtatt cgccgccccc cccatcttgc agaaggagca ctcctcggag agcaagaaca
     7561 ccaatgtgct cagtgcggaa aagcgggtcc agtccatcac caatggcagt gtcgtgagta
     7621 tttgctactc cctcagcctg gccgtcgatc ccgagtattc gtccgatggc gaaagcccca
     7681 gggaggataa tgaaagtaac gaggagatgg aaagtgaata cagcgccaac tgtgagtcga
     7741 gtgtggagct gattgagtcg aacgaggaga tcgagttcga ggtcggcacc ggcagcatga
     7801 acccgcacat tgagagcgag gtgacccaaa tgaccgttgg tgagtacgcc agctttcgca
     7861 tgacaccgcc agacgcggca gaggcactga tcctggcagt gggcagcgac accgtgcgaa
     7921 tccgctccct gctgagcgag aggccatgcc tcaattacaa catcctgctg ctgggcgtta
     7981 agggacccag tgaggagcgc atggaggccg ccttcttcaa gccccctctc tcgaagcaac
     8041 gagtggagta tgccctgaag catattcgag agtcgagtgc atcgacgctg gtggattttg
     8101 gctgtggctc cggaagtctg ttggatagtc tcctggacta cccaacaagc ctgcaaacga
     8161 ttataggcgt ggacatctcg cctaagggcc tcgcccgtgc cgcgaagatg ctgcacgtta
     8221 aattgaacaa ggaggcgtgc aatgttaaga gtgctactct gtacgatgga agcattctcg
     8281 agttcgacag ccgcctgcac gatgttgaca ttggcacttg tttggcagtg attgcagcca
     8341 tggaagagga tcaagcctgc gagtttggtg aaaaggtgtt gagtttgttc cacccgaagc
     8401 tgctcatagt gagcaccccc aattatgaat ttaacacgat actgcagcgc tcgacgcccg
     8461 agacgcagga ggagaacaac tccgagccac agctcccgaa attcaggaac catgacgcta
     8521 aattcgagtg gacacgggag cagtttaatc aatgggcttc gaagctgggc aagcgccaca
     8581 actactccgt ggagttcagc ggtgtcggag gcagtggcga ggtggaaccg ggctttgcca
     8641 gccagatcgc tatattccgg cgtgaagcta gctcggtcga gaatgtcgca gaaagtagca
     8701 tgcaacccta caaagtgatt tgggaatgga agaaggagga cgtcgaaaaa aagaagactg
     8761 acttgtaaaa gggtgggcgc gccgacccag ctttct
//
