LOCUS       sequence-211056-        8497 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 8497)
  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 8497)
  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..8497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..427
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             446..511
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1778..1789
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1801..1922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2632..2649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2803..3391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2883..2902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3562..4353,4354..4422))
                     /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     4185..4204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4423..4527)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4595..4613
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6010..6289
                     /label=copia promoter
                     /note="strong promoter from the Drosophila transposable 
                     element copia (Sinclair et al., 1986)"
     CDS             6335..7360
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6335..7360
                     /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          7723..7788
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             7918..7938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    8363..8497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 aattcgttgc aggacaggat gtggtgcccg atgtgactag ctctttgctg caggccgtcc
       61 tatcctctgg ttccgataag agacccagaa ctccggcccc ccaccgccca ccgccacccc
      121 catacatatg tggtacgcaa gtaagagtgc ctgcgcatgc cccatgtgcc ccaccaagag
      181 ctttgcatcc catacaagtc cccaaagtgg agaaccgaac caattcttcg cgggcagaac
      241 aaaagcttct gcacacgtct ccactcgaat ttggagccgg ccggcgtgtg caaaagaggt
      301 gaatcgaacg aaagacccgt gtgtaaagcc gcgtttccaa aatgtataaa accgagagca
      361 tctggccaat gtgcatcagt tgtggtcagc agcaaaatca agtgaatcat ctcagtgcaa
      421 ctaaaggggg gatctcgaga ccatggacta caaagaccat gacggtgatt ataaagatca
      481 tgacatcgat tacaaggatg acgatgacaa gaccggtatg gccgacaatg acgatctttt
      541 ggactacgag gatgaggagc agaccgagac cactgcggtc gaaaaccagg aggcccccaa
      601 gaaggatgtc aagggcacct atgtgtccat tcacagttcc ggcttccgcg atttcctcct
      661 gaaaccggag atcctgcgcg ccatcgtgga ctgcggcttc gagcatccct cggaggttca
      721 gcacgagtgc attccgcagg ccgtactggg catggacatc ctctgtcagg ccaagtccgg
      781 catgggtaac accgccgtct tcgttctggc cacgctgcag cagctggagc cgtcggacaa
      841 caacacctgc cacgtcctgg tcatgtgcca cacccgcgag ctggccttcc agatcagcaa
      901 ggagtatgag cgattctcca agtacatgcc cacagtcaag gtggctgtct tctttggcgg
      961 aatggctatt caaaaggacg aggagaccct caagagcggc accccgcata ttgtggtggg
     1021 cacccctggc cgaattctcg ccctcattcg caacaagaaa cttaatctga agcttttgaa
     1081 gcactttgtg ctcgacgagt gcgacaagat gctggagcag ctggatatgc gtcgtgacgt
     1141 tcaagagatt ttccgtagca cgccgcacgg caaacaagtg atgatgttct ctgccacatt
     1201 gagcaaggac attcgtcccg tttgcaaaaa gttcatgcaa gatcccatgg aggtctacgt
     1261 cgacgatgag gccaagctga cgctgcacgg actgcagcag cactacgtca atctgaagga
     1321 gaacgagaag aacaagaaac tgttcgaact gctcgacgtg ctcgagttca atcaggtggt
     1381 catctttgtg aagtctgtgc aacgttgcgt ggctctgtcg cagctgctga cggagcagaa
     1441 cttccccgcc atcggcatcc atcgtgggat gacccaggag gagcgtctga atcgctacca
     1501 gcagttcaag gacttccaga agcgcattct ggtggccacc aatctctttg gccgcggcat
     1561 ggacatcgag cgtgtgaaca tcgtgttcaa ctacgacatg cccgaggatt ccgacaccta
     1621 cttgcatcgc gtggcccgtg ccggtcgctt cggcaccaag ggactggcaa tcacattcgt
     1681 ttcggacgag aacgacgcca agatacttaa cgaagtacag gatcgtttcg atgtgaacat
     1741 cagtgagctg cccgaggaaa tcgatctctc tacatacatt gagggacgct aggcggccgc
     1801 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1861 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1921 taactagtgg atccactagg ggccgccgac gcgaggctgg atggccttcc ccattatgat
     1981 tcttctcgct tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt
     2041 agatgacgac catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc
     2101 gatcattgga ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg
     2161 gttggcatgg attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg
     2221 tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc
     2281 accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac
     2341 ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg
     2401 ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     2461 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     2521 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     2581 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc tcttccgctt cctcgctcac
     2641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3241 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3841 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     3901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     4201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     4561 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     4621 tcttcaagaa ttcacatttg tacgaatttt ttttttatca aaagttcgag tttttcacca
     4681 atttcctcat caaccgagca aggcaaacgg ctttgaataa tatggtgtta tatatacata
     4741 tatcaaatcg ctgctgactg cgtgattgat ggccccaaga ttacatatta tcgaatcagg
     4801 attcagaagg agatcaatgt caaatgcgga caggaacatg aaagacgcct gttatgcgca
     4861 attaaaaatt tgggtttaat tgctgtggaa actgttgttg gcggcatctt aagttcctgt
     4921 ttaacaacat caactactta tgtacgtaga agcgtttaag ccatttgcat acagatgaga
     4981 actggctttt gtgctaatca gtcaagatga ctccgatgat gatgactcat tacctgacca
     5041 gttttcgctg ctttcttttc aacaactact tgtatatgta ttgtatccaa tagcaataca
     5101 ttgaatttcc atggtctagt cacgtattat catttaattg acaccaagtc gtgttattgt
     5161 tgagctatcg agttcagctc aaacatttct tattcccatg aataagccgg caaaaatatg
     5221 caatctatga aagttaatat aagcaaacct tactttgact caataccaat gcactttgtg
     5281 tcgataggtt cacgcaattg aggcgattat tccgataacc caagcgattg actgttcccg
     5341 tttcgattcc aattgaaatt tggaaatgta caatagtttt gctatatgct gtcaagtacg
     5401 ctcttatctt ctctgggttt tcttcagagt ttcgaaacgc ttcttctttt ttttgttttt
     5461 tttttttttg gaatctcgta ttttggaagg ggctcccctc tggaatttgt tacactgtcg
     5521 ttatcattgc gaacaagcgg cccgaagcta tcagcgactt taacatttac aatgcacttt
     5581 tttacgacca attaaatgta cattttcctt tcttcgcccg ttgataagcg aacgcgatgt
     5641 ggcgcaggca atgtgttgct cttgcgacac aaacgcaatc aaaatggatt caatttcgct
     5701 ttttcccagt gaaacgaaga acgaaccgac catcatgata tgctcctctg catgttgcgt
     5761 attgaatcaa tgacaatttc aattaagccg cccgttcgtc atgcgttttc gtgcgcttcg
     5821 aaatgctgat aacgctgctg tcctccaact gctttgcatg tggacacaat tccatttatt
     5881 taattctttt atttggatcg gttaaattaa aaagcgcctt gttacgcatt taacgttgtt
     5941 tccggtgcgt ggtggtttca tgcttctggg aacggcaaat gggtttagga ttgggaaccc
     6001 ctcatcatct gttggaatat actattcaac ctacaaaagt aacgttaaac aacactactt
     6061 tatatttgat atgaatggcc acacctttta tgccataaaa catattgtaa gagaatacca
     6121 ctctttttat tccttctttc cttcttgtac gttttttgct gtaagtaggt cgtggtgctg
     6181 gtgttgcagt tgaaataact taaaatataa atcataaaac tcaaacataa acttgactat
     6241 ttatttattt attaagaaag gaaatataaa ttataaatta caacaggtta tggacctgca
     6301 gccaagcttg gcgctcgtcc gggggcaatg agatatgaaa aagcctgaac tcaccgcgac
     6361 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     6421 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     6481 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     6541 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     6601 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     6661 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     6721 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     6781 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     6841 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     6901 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     6961 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     7021 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     7081 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     7141 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     7201 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     7261 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     7321 aaaccgacgc cccagcactc gtccgagggc aaaggaatag agtagatgcc gaccgaacaa
     7381 gagctgattt cgagaacgcc tcagccagca actcgcgcga gcctagcaag gcaaatgcga
     7441 gagaacggcc ttacgcttgg tggcacagtt ctcgtccaca gttcgctaag ctcgctcggc
     7501 tgggtcgcgg gagggccggt cgcagtgatt caggcccttc tggattgtgt tggtccccag
     7561 ggcacgattg tcatgcccac gcactcgggt gatctgactg atcccgcaga ttggagatcg
     7621 ccgcccgtgc ctgccgattg ggtgcagatc tttgtgaagg aaccttactt ctgtggtgtg
     7681 acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta
     7741 agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat
     7801 ggaactgatg aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca
     7861 gaagaaatgc catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca
     7921 aaaaagaaga gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg
     7981 agtcatgctg tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa
     8041 aaagctgcac tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt tataagtagg
     8101 cataacagtt ataatcataa catactgttt tttcttactc cacacaggca tagagtgtct
     8161 gctattaata actatgctca aaaattgtgt acctttagct ttttaatttg taaaggggtt
     8221 aataaggaat atttgatgta tagtgccttg actagagatc ataatcagcc ataccacatt
     8281 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     8341 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     8401 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     8461 gtccaaactc atcaatgtat cttatcatgt ctggatc
//
