LOCUS       sequence-373457-        9121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin-selectable expression of HA-tagged truncated Dora 
            (CG34401) in Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9121)
  AUTHORS   Kingston ER, Blodgett LW, Bartel DP
  TITLE     Endogenous transcripts direct microRNA degradation in Drosophila, 
            and this targeted degradation is required for proper embryonic 
            development.
  JOURNAL   Mol Cell. 2022 Sep 15. pii: S1097-2765(22)00849-8. doi: 
            10.1016/j.molcel.2022.08.029.
  PUBMED    36150386
REFERENCE   2  (bases 1 to 9121)
  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: "Mol Cell. 
            2022 Sep 15. pii: S1097-2765(22)00849-8. doi: 
            10.1016/j.molcel.2022.08.029."
COMMENT     Sequence Label: HA-dora[A]_rescue_construct
FEATURES             Location/Qualifiers
     source          1..9121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1723..1749
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1762..1788
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1801..1827
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4765..4818
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4843..5442
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4843..4862)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5339..5359
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5450..5584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5474..5493)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5528..5547
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(6185..6773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6265..6284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6944..7735,7736..7804))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7567..7586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7805..7909)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7977..7995
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(8147..8222)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg cagcggaagg
       61 aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg acgttggagc
      121 cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc gccggttgtt
      181 tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc tcaacgcacc
      241 gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt atttaaacaa
      301 agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact tattgccatg
      361 tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc tctttcactt
      421 tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat tacgctgaca
      481 aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg catttgcaat
      541 aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca cacaaaaaaa
      601 aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt tagggtaaca
      661 acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag agagagagag
      721 aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct ctctcttcgt
      781 ctctgttgcg caaacgcata gactgcactg aaaaaatcga ttacctattt tttatgaatg
      841 aatatttgca ctattactat tcaaaactat taagatagca atcacattca atagccaaat
      901 actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc tgcatattta
      961 ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct ggcagctgtt
     1021 tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta gtgaatgccc
     1081 tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa gtgcagatag
     1141 cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat cgctggacat
     1201 tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac aatgagtagt
     1261 tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac cagctgaagt
     1321 atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga cccatggaag
     1381 tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt tgcggaggaa
     1441 attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg gtaaaagttt
     1501 tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc taagcgggct
     1561 ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc ttgtgctgtg
     1621 tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa tccagagacc
     1681 ccggatcggg gtacctacta gtccagtgtg gtggaattca tgtatccata tgatgttcca
     1741 gattatgctg gaggttcttc atacccctat gatgtgcctg actacgctgg cagcgccgga
     1801 tacccttatg atgtgcctga ttatgctgga gggagcggcg gaagtgggag ttcaggaggc
     1861 tccggatcaa gcggcagtag tggtggaagc gaccgcttca gcttcgacga ctccatccgc
     1921 ttcgaggaag actccctctg cagctggagc tctgaaccgg aatcgctgtg caacaactgg
     1981 cgcggctgga agaagcccgc tgcaggcagc ggcgcaggca atggcggctt gagcggcatg
     2041 ggcggtatgg ctgtggccgg cggctcagga gcaccgccgt tcggaggagc aggagcctct
     2101 ggttcctgca caccgggcgg caatggaacg gccatggcca acacatccac ctgcggcaga
     2161 cattatgaag tatccacatt ggccgaactg gcagcccgct gtgtggcctc ctacataccg
     2221 ttcgagctgg ttgagcacgt gtatccgccg gtgccggagc agctgcagct gcgcattgcg
     2281 ttctggagct ttccggacaa cgaggaggac atccggctct actcctgtct ggccaatagc
     2341 tcggcggacg agttcaatcg cggcgactcg ttgtttcggt tgcgagctgt caaggatccg
     2401 ctgcagatag gctttcacct ctcggccagc gttgtcaatc agacgccgcg tgcctatttc
     2461 aatgtggccg tcacctttga ccgccgacgc atctcctctt gcaactgcac ctgcacctcg
     2521 tccgcctact ggtgctccca cgtagtcgcc gtttgtctgc atcgcatcca ttgcccccag
     2581 gaggtctgtc tgcgtgcccc ggtatcggaa tcattgactc gcctgcaacg cgaccaattg
     2641 caaaagtttg cccagtatct gatcagcgag ctgcctcagc agatcttgcc gacggctcag
     2701 cggctcctgg atgagttgct cagtgcgcaa cccacggcga tcaacactgt gtgcggtgca
     2761 cctgatccca cggctggggc ctcgatcaac gaccagacca gctggtatct ggacgagaag
     2821 acgttgcata acaacattaa acgaatcctc atcaagttct gtttgcccgc cccgattgtg
     2881 tttagcgacg taaactatct gacgaactcg gctcccccag cggcggccga atggagctcg
     2941 ctactccgac cgctgcgggg acgtgagccg gagggcatgt ggaatctact ttcgatcgta
     3001 cgcgagatgt acaggcgctg tgatcggaac gcagtgcgtc tgctggagat catcacggag
     3061 gagtgcttgt actgcgatca gatactcatc tggtggttcc agacgaagtt ggcactgatg
     3121 atggggtccc acggccactc cggcggtaag cattctaaca ctcactccaa ctccacggca
     3181 ctgcagcatg cttgcagttc gctgtgcgac gaaatcgtgg cactgtggcg attggcagcg
     3241 ctcaatcccg gcctggcgcc ggatgagcga gatatgctgc acgctcagtt cacggcctgg
     3301 catctaaaga ttcttgaccg ggtggtcaag agccgaatga tgccgtcgta caccaacaag
     3361 caccagcaga actcgcgatc cgagacggag ctgtttatcg gcttcaagcc ggccatcgag
     3421 gcgtgctact tggactggga gggctacccc ataccgggag tgacgcacac ccacgacacg
     3481 aatcccatct actactcgcc gttcacttgc ttcaagcaca cggacctcaa gggcgagtct
     3541 aacaatcccg gccagctgaa tgccacccaa gcgctgatgt cgaacaacaa gcactacaat
     3601 tactttagtt catccagcga tcatggcatg cacgcaggag ccttcaagca gaggttggac
     3661 cggccttttc gtgagtcgcg attctatacg aatccggccg atctggacgg aggagcagcg
     3721 ttggcgggcg gaggatccgg taggttttcc acgggactgg gctccgttgc cgttggagga
     3781 gctggtgcag gaggcttggg tcagatgagc ggaggagtta acaactctgt tggaggatca
     3841 gcggccgccg aagtgaatcc cgtgcagcag cagcttccca gtggctcggc gggagtcggt
     3901 gcctctgtga caaatgccaa ggttgtggta gcaacagatg gcaatcgttc gagtgccagc
     3961 agcgagggat tctgcgagaa cgacgatttc ggcggcgaca ccagcagcag tcacaactac
     4021 tgtccacctg gacaggcggt tgtgcctggc cagaaatcag ccctcacgga gtcggattcg
     4081 cagagcagct tcgatgcagt gtcgcatcag agcaaggatg agccaccagt cgggggagtg
     4141 ggtgtggcag tgggtgttga acaagccctt tccacatctg acacttcgtc ggcctcctcc
     4201 tcatcgtcat ctgcttctac agcttcaggg agctctaata gttccacttc ctctatgctt
     4261 atggctggac aggaggctac tgtcggtgtt ggtggcgctg tgcagcaaac aactcgtcgc
     4321 ctcagcaagg atgaatcctt tagtagcagt agcgacgagt tcaaccaggg aggcgtgggt
     4381 ggatcagcag gtcgagtggc tatggcctcc agtggaagcg gatcgggtgc tggcgacaca
     4441 gtagcggtgg gatcgggatc aacagctggc ggagatctga cgccggtgcc cagcacgtcg
     4501 gcagctgccc gtgctgcctt ggccgccagc tcgacagggc cggtgatttc tgcttcacct
     4561 catctcgcag ctagcgccgc tggaggctta ggagtgctgg gtgtgggtgc agaccagcct
     4621 tgcacctcat ccgcgcatgc agcccgagca ctggcagctg cagtggctgc ttccagcgat
     4681 aagccgcatg ttttctccaa tgtccggccc acagaggacg ccaaggccgg ccaggcaaaa
     4741 aagaaaaagg gatcaggcaa gcttgagggc agaggaagtc ttctaacatg cggtgacgtg
     4801 gaggagaatc ccggccctgc tagcggtagc ggcagcggta gcatgaccga gtacaagccc
     4861 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
     4921 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
     4981 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     5041 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     5101 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     5161 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     5221 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     5281 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     5341 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     5401 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gactcgaggg atccagacat
     5461 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     5521 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     5581 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     5641 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc agtcgaccga
     5701 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     5761 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     5821 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     5881 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     5941 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg
     6001 tcttgctggc gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt
     6061 ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
     6121 atcagggaca gcttcaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6361 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6601 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     6661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     7261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7561 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7921 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     7981 ggcgtatcac gaggcccttt cgtcttcaag aattaattct atattctaaa aacacaaatg
     8041 atacttctaa aaaaaaatca tgaatggcat caactctgaa tcaaatcttt gcagatgcac
     8101 ctacttctca tttccactgt cacatcattt ttccagatct aaaaaagcac cgactcggtg
     8161 ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa
     8221 actacataac gaataaaaaa ggatttaagt gcaaatggta aaaaagtgtc gagtttttct
     8281 tgagttgatt gtgctgtaat gagactctgc attcggcatt tgactcggct tttcctactc
     8341 gtgccgtatt tcaggctgca agtcgaacag atctcgctgc ctgttatgtg gcccacaaac
     8401 caagacacgt tttatggcca ttaaagctgg ctgatcgtcg ccaaacacca aatacataat
     8461 gaatatgtac acattcgaga aagaagcgat caaagaagcg tcttcgggcg gagtaggaga
     8521 atgcggagga gaaggagaac gagctgatct agtatctctc cacaatccaa tgccaactga
     8581 ccaactggcc atattcggag caatttgaag ccaatttcca tcgcctggcg atcgctccat
     8641 tcttggctat atgtttttca ccgttacccg gggccatttt caaagactcg tcggcaagat
     8701 aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga atgctgagga atttcgcgat
     8761 gacgtcggcg agtattttga agaatgagaa taatttgtat ttatacgaaa atcagttagt
     8821 ggaattttct acaaaaacat gttatctata gataattttg ttgcaaaata tgttgactat
     8881 gacaaagatt gtatgtatat ctttaatgta ttctcatttt cttatgtatt tataatggca
     8941 atgatgatac tgatgatatt ttaagatgat gccagaccaa aaggcttgaa tttctgcgtc
     9001 ttttgccgaa cgcagtgcat gtgcaattgt tgttttttgg aatattcaat tttcggactg
     9061 tccgctttga tttcagtttc ttggcttatt caaaaagcaa agtaaagcca aaaaagcgag
     9121 a
//
