LOCUS       sequence-207168-        8496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flag-tagged D. melanogaster Hel25E with K91N mutation that
            greatly reduces helicase activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8496)
  AUTHORS   Huang C, Liang D, Tatomer DC, Wilusz JE
  TITLE     A length-dependent evolutionarily conserved pathway controls nuclear
            export of circular RNAs.
  JOURNAL   Genes Dev. 2018 May 17. pii: gad.314856.118. doi: 
            10.1101/gad.314856.118.
  PUBMED    29773557
REFERENCE   2  (bases 1 to 8496)
  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: "Genes Dev. 
            2018 May 17. pii: gad.314856.118. doi: 10.1101/gad.314856.118."
COMMENT     Sequence Label: Hy_pMT Flag-Hel25E K91N
FEATURES             Location/Qualifiers
     source          1..8496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1031..1049
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2445..2724
                     /label=copia promoter
                     /note="strong promoter from the Drosophila transposable 
                     element copia (Sinclair et al., 1986)"
     CDS             2770..3795
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2770..3795
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          4158..4223
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4353..4373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4798..4932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4933..5359
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             5378..5443
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6710..6721
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6733..6854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7564..7581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7735..8323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7815..7834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8494..8496,1..789,790..858))
                     /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"
ORIGIN
        1 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     1021 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattca catttgtacg
     1081 aatttttttt ttatcaaaag ttcgagtttt tcaccaattt cctcatcaac cgagcaaggc
     1141 aaacggcttt gaataatatg gtgttatata tacatatatc aaatcgctgc tgactgcgtg
     1201 attgatggcc ccaagattac atattatcga atcaggattc agaaggagat caatgtcaaa
     1261 tgcggacagg aacatgaaag acgcctgtta tgcgcaatta aaaatttggg tttaattgct
     1321 gtggaaactg ttgttggcgg catcttaagt tcctgtttaa caacatcaac tacttatgta
     1381 cgtagaagcg tttaagccat ttgcatacag atgagaactg gcttttgtgc taatcagtca
     1441 agatgactcc gatgatgatg actcattacc tgaccagttt tcgctgcttt cttttcaaca
     1501 actacttgta tatgtattgt atccaatagc aatacattga atttccatgg tctagtcacg
     1561 tattatcatt taattgacac caagtcgtgt tattgttgag ctatcgagtt cagctcaaac
     1621 atttcttatt cccatgaata agccggcaaa aatatgcaat ctatgaaagt taatataagc
     1681 aaaccttact ttgactcaat accaatgcac tttgtgtcga taggttcacg caattgaggc
     1741 gattattccg ataacccaag cgattgactg ttcccgtttc gattccaatt gaaatttgga
     1801 aatgtacaat agttttgcta tatgctgtca agtacgctct tatcttctct gggttttctt
     1861 cagagtttcg aaacgcttct tctttttttt gttttttttt ttttggaatc tcgtattttg
     1921 gaaggggctc ccctctggaa tttgttacac tgtcgttatc attgcgaaca agcggcccga
     1981 agctatcagc gactttaaca tttacaatgc acttttttac gaccaattaa atgtacattt
     2041 tcctttcttc gcccgttgat aagcgaacgc gatgtggcgc aggcaatgtg ttgctcttgc
     2101 gacacaaacg caatcaaaat ggattcaatt tcgctttttc ccagtgaaac gaagaacgaa
     2161 ccgaccatca tgatatgctc ctctgcatgt tgcgtattga atcaatgaca atttcaatta
     2221 agccgcccgt tcgtcatgcg ttttcgtgcg cttcgaaatg ctgataacgc tgctgtcctc
     2281 caactgcttt gcatgtggac acaattccat ttatttaatt cttttatttg gatcggttaa
     2341 attaaaaagc gccttgttac gcatttaacg ttgtttccgg tgcgtggtgg tttcatgctt
     2401 ctgggaacgg caaatgggtt taggattggg aacccctcat catctgttgg aatatactat
     2461 tcaacctaca aaagtaacgt taaacaacac tactttatat ttgatatgaa tggccacacc
     2521 ttttatgcca taaaacatat tgtaagagaa taccactctt tttattcctt ctttccttct
     2581 tgtacgtttt ttgctgtaag taggtcgtgg tgctggtgtt gcagttgaaa taacttaaaa
     2641 tataaatcat aaaactcaaa cataaacttg actatttatt tatttattaa gaaaggaaat
     2701 ataaattata aattacaaca ggttatggac ctgcagccaa gcttggcgct cgtccggggg
     2761 caatgagata tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2821 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2881 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2941 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     3001 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     3061 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     3121 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     3181 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     3241 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     3301 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3361 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3421 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3481 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3541 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3601 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3661 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3721 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3781 agggcaaagg aatagagtag atgccgaccg aacaagagct gatttcgaga acgcctcagc
     3841 cagcaactcg cgcgagccta gcaaggcaaa tgcgagagaa cggccttacg cttggtggca
     3901 cagttctcgt ccacagttcg ctaagctcgc tcggctgggt cgcgggaggg ccggtcgcag
     3961 tgattcaggc ccttctggat tgtgttggtc cccagggcac gattgtcatg cccacgcact
     4021 cgggtgatct gactgatccc gcagattgga gatcgccgcc cgtgcctgcc gattgggtgc
     4081 agatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag
     4141 agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga
     4201 ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt
     4261 ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg
     4321 aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag gtagaagacc
     4381 ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa
     4441 ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa
     4501 ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat cataacatac
     4561 tgttttttct tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat
     4621 tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg
     4681 ccttgactag agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     4741 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     4801 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4861 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4921 catgtctgga tcaattcgtt gcaggacagg atgtggtgcc cgatgtgact agctctttgc
     4981 tgcaggccgt cctatcctct ggttccgata agagacccag aactccggcc ccccaccgcc
     5041 caccgccacc cccatacata tgtggtacgc aagtaagagt gcctgcgcat gccccatgtg
     5101 ccccaccaag agctttgcat cccatacaag tccccaaagt ggagaaccga accaattctt
     5161 cgcgggcaga acaaaagctt ctgcacacgt ctccactcga atttggagcc ggccggcgtg
     5221 tgcaaaagag gtgaatcgaa cgaaagaccc gtgtgtaaag ccgcgtttcc aaaatgtata
     5281 aaaccgagag catctggcca atgtgcatca gttgtggtca gcagcaaaat caagtgaatc
     5341 atctcagtgc aactaaaggg gggatctcga gaccatggac tacaaagacc atgacggtga
     5401 ttataaagat catgacatcg attacaagga tgacgatgac aagaccggta tggccgacaa
     5461 tgacgatctt ttggactacg aggatgagga gcagaccgag accactgcgg tcgaaaacca
     5521 ggaggccccc aagaaggatg tcaagggcac ctatgtgtcc attcacagtt ccggcttccg
     5581 cgatttcctc ctgaaaccgg agatcctgcg cgccatcgtg gactgcggct tcgagcatcc
     5641 ctcggaggtt cagcacgagt gcattccgca ggccgtactg ggcatggaca tcctctgtca
     5701 ggccaagtcc ggcatgggta acaccgccgt cttcgttctg gccacgctgc agcagctgga
     5761 gccgtcggac aacaacacct gccacgtcct ggtcatgtgc cacacccgcg agctggcctt
     5821 ccagatcagc aaggagtatg agcgattctc caagtacatg cccacagtca aggtggctgt
     5881 cttctttggc ggaatggcta ttcaaaagga cgaggagacc ctcaagagcg gcaccccgca
     5941 tattgtggtg ggcacccctg gccgaattct cgccctcatt cgcaacaaga aacttaatct
     6001 gaagcttttg aagcactttg tgctcgacga gtgcgacaag atgctggagc agctggatat
     6061 gcgtcgtgac gttcaagaga ttttccgtag cacgccgcac ggcaaacaag tgatgatgtt
     6121 ctctgccaca ttgagcaagg acattcgtcc cgtttgcaaa aagttcatgc aagatcccat
     6181 ggaggtctac gtcgacgatg aggccaagct gacgctgcac ggactgcagc agcactacgt
     6241 caatctgaag gagaacgaga agaacaagaa actgttcgaa ctgctcgacg tgctcgagtt
     6301 caatcaggtg gtcatctttg tgaagtctgt gcaacgttgc gtggctctgt cgcagctgct
     6361 gacggagcag aacttccccg ccatcggcat ccatcgtggg atgacccagg aggagcgtct
     6421 gaatcgctac cagcagttca aggacttcca gaagcgcatt ctggtggcca ccaatctctt
     6481 tggccgcggc atggacatcg agcgtgtgaa catcgtgttc aactacgaca tgcccgagga
     6541 ttccgacacc tacttgcatc gcgtggcccg tgccggtcgc ttcggcacca agggactggc
     6601 aatcacattc gtttcggacg agaacgacgc caagatactt aacgaagtac aggatcgttt
     6661 cgatgtgaac atcagtgagc tgcccgagga aatcgatctc tctacataca ttgagggacg
     6721 ctaggcggcc gcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6781 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6841 atcaatgtat cttaactagt ggatccacta ggggccgccg acgcgaggct ggatggcctt
     6901 ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct
     6961 gtccaggcag gtagatgacg accatcaggg acagcttcaa ggatcgctcg cggctcttac
     7021 cagcctaact tcgatcattg gaccgctgat cgtcacggcg atttatgccg cctcggcgag
     7081 cacatggaac gggttggcat ggattgtagg cgccgcccta taccttgtct gcctccccgc
     7141 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     7201 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     7261 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     7321 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     7381 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     7441 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     7501 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gctcttccgc
     7561 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     7621 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     7681 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     7741 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7801 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7861 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7921 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7981 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     8041 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     8101 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     8161 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     8221 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     8281 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     8341 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     8401 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     8461 ctaaagtata tatgagtaaa cttggtctga cagtta
//
