LOCUS       sequence-373462-        8953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin-selectable expression of truncated Dora (CG34401) in 
            Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8953)
  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 8953)
  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: dora[A]_rescue_construct
FEATURES             Location/Qualifiers
     source          1..8953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4541..4594
                     /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             4619..5218
                     /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(4619..4638)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5115..5135
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5226..5360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5250..5269)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5304..5323
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5961..6549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6041..6060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6720..7511,7512..7580))
                     /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     7343..7362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7581..7685)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7753..7771
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(7923..7998)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aaggaagggt ggggcggggc gtggcggggt ctgtggctgg gcgcgacgtc accgacgttg
       61 gagccactcc tttgaccatg tgtgcgtgtg tgtattattc gtgtctcgcc actcgccggt
      121 tgtttttttc tttttatgct gcgctctctc tagcgccatc tcgcttacgc atgctcaacg
      181 caccgcatgt tgccgtttcc ttttatgcgt cattttggct cgaaataggc aattatttaa
      241 acaaagatta gtcaacgaaa acgctaaaat aaataagtct acaatatggt tacttattgc
      301 catgtgtgtg cagccaacga tagcaacaaa agcaacaaca caggtggctt tccctctttc
      361 actttttgtt tgcaagccgc gtgcgagcaa gacggcacga ccggcaaacg caattacgct
      421 gacaaagagc agacgaagtt ttggcgaaaa acatcaaggc gcctgatacg aatgcatttg
      481 caataacaat tgcgatattt aatattgttt atgaagctgt ttgacttcaa aacacacaaa
      541 aaaaaaaata aaacaaatta tttgaaagag aattaggaat cggacgctta tcgttagggt
      601 aacaacaaga aatgcttact gagtcacagc ctctggaaaa ctgccgcaag ccagagagag
      661 agagaaaaag agggagagca gcttagaccg catgtgcttg tgtgtgaggc gtctctctct
      721 tcgtctctgt tgcgcaaacg catagactgc actgaaaaaa tcgattacct attttttatg
      781 aatgaatatt tgcactatta ctattcaaaa ctattaagat agcaatcaca ttcaatagcc
      841 aaatactata ccacctgagc gatgcaacga aatgatcaat ttgagcaaaa atgctgcata
      901 tttaggacgg catcattata gaaatgcttc ttgctgtgta cttttctctc gtctggcagc
      961 tgtttcgccg ttattgttaa aaccggctta agttaggtgt gttttctacg actagtgaat
     1021 gccctactag aagatgtgtg ttgcacaaaa tgtccctgga ataaccaatt tgaagtgcag
     1081 atagcagtaa acgtaagcta atatgaatat tatttaactg taatgtttta atatcgctgg
     1141 acattactaa taaacccact ataaacacat gtacatatgt atgttttggc atacaatgag
     1201 tagttgggga aaaaatgtgt aaaagcaccg tgaccatcac agcataaaga taaccagctg
     1261 aagtatcgaa tatgagtaac ccccaaattg aatcacatgc cgcaactgat aggacccatg
     1321 gaagtacact cttcatggcg atatacaaga cacacacaag cacgaacacc cagttgcgga
     1381 ggaaattctc cgtaaatgaa aacccaatcg gcgaacaatt catacccata tatggtaaaa
     1441 gttttgaacg cgacttgaga gcggagagca ttgcggctga taaggtttta gcgctaagcg
     1501 ggctttataa aacgggctgc gggaccagtt ttcatatcac taccgtttga gttcttgtgc
     1561 tgtgtggata ctcctcccga cacaaagccg ctccatcagc cagcagtcgt ctaatccaga
     1621 gaccccggat cggggtacct actagtccag tgtggtggaa ttcatggacc gcttcagctt
     1681 cgacgactcc atccgcttcg aggaagactc cctctgcagc tggagctctg aaccggaatc
     1741 gctgtgcaac aactggcgcg gctggaagaa gcccgctgca ggcagcggcg caggcaatgg
     1801 cggcttgagc ggcatgggcg gtatggctgt ggccggcggc tcaggagcac cgccgttcgg
     1861 aggagcagga gcctctggtt cctgcacacc gggcggcaat ggaacggcca tggccaacac
     1921 atccacctgc ggcagacatt atgaagtatc cacattggcc gaactggcag cccgctgtgt
     1981 ggcctcctac ataccgttcg agctggttga gcacgtgtat ccgccggtgc cggagcagct
     2041 gcagctgcgc attgcgttct ggagctttcc ggacaacgag gaggacatcc ggctctactc
     2101 ctgtctggcc aatagctcgg cggacgagtt caatcgcggc gactcgttgt ttcggttgcg
     2161 agctgtcaag gatccgctgc agataggctt tcacctctcg gccagcgttg tcaatcagac
     2221 gccgcgtgcc tatttcaatg tggccgtcac ctttgaccgc cgacgcatct cctcttgcaa
     2281 ctgcacctgc acctcgtccg cctactggtg ctcccacgta gtcgccgttt gtctgcatcg
     2341 catccattgc ccccaggagg tctgtctgcg tgccccggta tcggaatcat tgactcgcct
     2401 gcaacgcgac caattgcaaa agtttgccca gtatctgatc agcgagctgc ctcagcagat
     2461 cttgccgacg gctcagcggc tcctggatga gttgctcagt gcgcaaccca cggcgatcaa
     2521 cactgtgtgc ggtgcacctg atcccacggc tggggcctcg atcaacgacc agaccagctg
     2581 gtatctggac gagaagacgt tgcataacaa cattaaacga atcctcatca agttctgttt
     2641 gcccgccccg attgtgttta gcgacgtaaa ctatctgacg aactcggctc ccccagcggc
     2701 ggccgaatgg agctcgctac tccgaccgct gcggggacgt gagccggagg gcatgtggaa
     2761 tctactttcg atcgtacgcg agatgtacag gcgctgtgat cggaacgcag tgcgtctgct
     2821 ggagatcatc acggaggagt gcttgtactg cgatcagata ctcatctggt ggttccagac
     2881 gaagttggca ctgatgatgg ggtcccacgg ccactccggc ggtaagcatt ctaacactca
     2941 ctccaactcc acggcactgc agcatgcttg cagttcgctg tgcgacgaaa tcgtggcact
     3001 gtggcgattg gcagcgctca atcccggcct ggcgccggat gagcgagata tgctgcacgc
     3061 tcagttcacg gcctggcatc taaagattct tgaccgggtg gtcaagagcc gaatgatgcc
     3121 gtcgtacacc aacaagcacc agcagaactc gcgatccgag acggagctgt ttatcggctt
     3181 caagccggcc atcgaggcgt gctacttgga ctgggagggc taccccatac cgggagtgac
     3241 gcacacccac gacacgaatc ccatctacta ctcgccgttc acttgcttca agcacacgga
     3301 cctcaagggc gagtctaaca atcccggcca gctgaatgcc acccaagcgc tgatgtcgaa
     3361 caacaagcac tacaattact ttagttcatc cagcgatcat ggcatgcacg caggagcctt
     3421 caagcagagg ttggaccggc cttttcgtga gtcgcgattc tatacgaatc cggccgatct
     3481 ggacggagga gcagcgttgg cgggcggagg atccggtagg ttttccacgg gactgggctc
     3541 cgttgccgtt ggaggagctg gtgcaggagg cttgggtcag atgagcggag gagttaacaa
     3601 ctctgttgga ggatcagcgg ccgccgaagt gaatcccgtg cagcagcagc ttcccagtgg
     3661 ctcggcggga gtcggtgcct ctgtgacaaa tgccaaggtt gtggtagcaa cagatggcaa
     3721 tcgttcgagt gccagcagcg agggattctg cgagaacgac gatttcggcg gcgacaccag
     3781 cagcagtcac aactactgtc cacctggaca ggcggttgtg cctggccaga aatcagccct
     3841 cacggagtcg gattcgcaga gcagcttcga tgcagtgtcg catcagagca aggatgagcc
     3901 accagtcggg ggagtgggtg tggcagtggg tgttgaacaa gccctttcca catctgacac
     3961 ttcgtcggcc tcctcctcat cgtcatctgc ttctacagct tcagggagct ctaatagttc
     4021 cacttcctct atgcttatgg ctggacagga ggctactgtc ggtgttggtg gcgctgtgca
     4081 gcaaacaact cgtcgcctca gcaaggatga atcctttagt agcagtagcg acgagttcaa
     4141 ccagggaggc gtgggtggat cagcaggtcg agtggctatg gcctccagtg gaagcggatc
     4201 gggtgctggc gacacagtag cggtgggatc gggatcaaca gctggcggag atctgacgcc
     4261 ggtgcccagc acgtcggcag ctgcccgtgc tgccttggcc gccagctcga cagggccggt
     4321 gatttctgct tcacctcatc tcgcagctag cgccgctgga ggcttaggag tgctgggtgt
     4381 gggtgcagac cagccttgca cctcatccgc gcatgcagcc cgagcactgg cagctgcagt
     4441 ggctgcttcc agcgataagc cgcatgtttt ctccaatgtc cggcccacag aggacgccaa
     4501 ggccggccag gcaaaaaaga aaaagggatc aggcaagctt gagggcagag gaagtcttct
     4561 aacatgcggt gacgtggagg agaatcccgg ccctgctagc ggtagcggca gcggtagcat
     4621 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     4681 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     4741 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     4801 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     4861 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     4921 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     4981 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     5041 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     5101 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5161 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgact
     5221 cgagggatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     5281 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     5341 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     5401 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     5461 atgatcagtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     5521 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     5581 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     5641 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     5701 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     5761 gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc cttccccatt
     5821 atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat gctgtccagg
     5881 caggtagatg acgaccatca gggacagctt caaggccagc aaaaggccag gaaccgtaaa
     5941 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6001 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6061 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6121 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6181 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6241 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6301 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6361 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6421 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6481 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6541 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6601 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6661 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6721 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6781 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6841 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6901 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6961 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7021 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7081 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7141 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7201 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7261 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7321 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7381 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7441 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7501 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7561 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7621 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7681 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     7741 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt aattctatat
     7801 tctaaaaaca caaatgatac ttctaaaaaa aaatcatgaa tggcatcaac tctgaatcaa
     7861 atctttgcag atgcacctac ttctcatttc cactgtcaca tcatttttcc agatctaaaa
     7921 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     7981 tatttctagc tctaaaacta cataacgaat aaaaaaggat ttaagtgcaa atggtaaaaa
     8041 agtgtcgagt ttttcttgag ttgattgtgc tgtaatgaga ctctgcattc ggcatttgac
     8101 tcggcttttc ctactcgtgc cgtatttcag gctgcaagtc gaacagatct cgctgcctgt
     8161 tatgtggccc acaaaccaag acacgtttta tggccattaa agctggctga tcgtcgccaa
     8221 acaccaaata cataatgaat atgtacacat tcgagaaaga agcgatcaaa gaagcgtctt
     8281 cgggcggagt aggagaatgc ggaggagaag gagaacgagc tgatctagta tctctccaca
     8341 atccaatgcc aactgaccaa ctggccatat tcggagcaat ttgaagccaa tttccatcgc
     8401 ctggcgatcg ctccattctt ggctatatgt ttttcaccgt tacccggggc cattttcaaa
     8461 gactcgtcgg caagataaga ttgtgtcact cgctgtctct cttcatttgt cgaagaatgc
     8521 tgaggaattt cgcgatgacg tcggcgagta ttttgaagaa tgagaataat ttgtatttat
     8581 acgaaaatca gttagtggaa ttttctacaa aaacatgtta tctatagata attttgttgc
     8641 aaaatatgtt gactatgaca aagattgtat gtatatcttt aatgtattct cattttctta
     8701 tgtatttata atggcaatga tgatactgat gatattttaa gatgatgcca gaccaaaagg
     8761 cttgaatttc tgcgtctttt gccgaacgca gtgcatgtgc aattgttgtt ttttggaata
     8821 ttcaattttc ggactgtccg ctttgatttc agtttcttgg cttattcaaa aagcaaagta
     8881 aagccaaaaa agcgagatgg caataccaaa tgcggcaaaa cggtagtgga aggaaagggg
     8941 tgcggggcag cgg
//
