LOCUS       Exported                8496 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Flag-tagged D. melanogaster Hel25E with D193E mutation 
            that greatly reduces ATP binding affinity.
ACCESSION   .
VERSION     .
KEYWORDS    Hy_pMT Flag-Hel25E D193E
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8496
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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(3562..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        6009..6288
                     /label=copia promoter
                     /note="strong promoter from the Drosophila transposable 
                     element copia (Sinclair et al., 1986)"
     CDS             6334..7359
                     /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          7722..7787
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             7917..7937
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    8362..8496
                     /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 catgggtaag 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 ctcgaagagt 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 ttttttttgg aatctcgtat tttggaaggg gctcccctct ggaatttgtt acactgtcgt
     5521 tatcattgcg aacaagcggc ccgaagctat cagcgacttt aacatttaca atgcactttt
     5581 ttacgaccaa ttaaatgtac attttccttt cttcgcccgt tgataagcga acgcgatgtg
     5641 gcgcaggcaa tgtgttgctc ttgcgacaca aacgcaatca aaatggattc aatttcgctt
     5701 tttcccagtg aaacgaagaa cgaaccgacc atcatgatat gctcctctgc atgttgcgta
     5761 ttgaatcaat gacaatttca attaagccgc ccgttcgtca tgcgttttcg tgcgcttcga
     5821 aatgctgata acgctgctgt cctccaactg ctttgcatgt ggacacaatt ccatttattt
     5881 aattctttta tttggatcgg ttaaattaaa aagcgccttg ttacgcattt aacgttgttt
     5941 ccggtgcgtg gtggtttcat gcttctggga acggcaaatg ggtttaggat tgggaacccc
     6001 tcatcatctg ttggaatata ctattcaacc tacaaaagta acgttaaaca acactacttt
     6061 atatttgata tgaatggcca caccttttat gccataaaac atattgtaag agaataccac
     6121 tctttttatt ccttctttcc ttcttgtacg ttttttgctg taagtaggtc gtggtgctgg
     6181 tgttgcagtt gaaataactt aaaatataaa tcataaaact caaacataaa cttgactatt
     6241 tatttattta ttaagaaagg aaatataaat tataaattac aacaggttat ggacctgcag
     6301 ccaagcttgg cgctcgtccg ggggcaatga gatatgaaaa agcctgaact caccgcgacg
     6361 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     6421 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     6481 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     6541 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     6601 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     6661 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     6721 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     6781 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     6841 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     6901 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     6961 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     7021 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     7081 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     7141 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     7201 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     7261 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     7321 aaccgacgcc ccagcactcg tccgagggca aaggaataga gtagatgccg accgaacaag
     7381 agctgatttc gagaacgcct cagccagcaa ctcgcgcgag cctagcaagg caaatgcgag
     7441 agaacggcct tacgcttggt ggcacagttc tcgtccacag ttcgctaagc tcgctcggct
     7501 gggtcgcggg agggccggtc gcagtgattc aggcccttct ggattgtgtt ggtccccagg
     7561 gcacgattgt catgcccacg cactcgggtg atctgactga tcccgcagat tggagatcgc
     7621 cgcccgtgcc tgccgattgg gtgcagatct ttgtgaagga accttacttc tgtggtgtga
     7681 cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa
     7741 gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg
     7801 gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag
     7861 aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa
     7921 aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga
     7981 gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa
     8041 aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc
     8101 ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg
     8161 ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta
     8221 ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt
     8281 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     8341 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     8401 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     8461 tccaaactca tcaatgtatc ttatcatgtc tggatc
//
