LOCUS       sequence-332013-        7440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For doxycycline inducible mammalian expression of EGFP tagged 
            Spastin M87 with exon 4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7440)
  AUTHORS   Cupido T, Pisa R, Kelley ME, Kapoor TM
  TITLE     Designing a chemical inhibitor for the AAA protein spastin using 
            active site mutations.
  JOURNAL   Nat Chem Biol. 2019 May;15(5):444-452. doi: 
            10.1038/s41589-019-0225-6. Epub 2019 Feb 18.
  PUBMED    30778202
REFERENCE   2  (bases 1 to 7440)
  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"; doi: 
            "10.1038/s41589-019-0225-6"; journalName: "Nat Chem Biol"; date: 
            "2019-05"; volume: "15"; issue: "5"; pages: "444-452"
COMMENT     Sequence Label: HA, EGFP Spastin M87 with exon 4
FEATURES             Location/Qualifiers
     source          1..7440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             762..788
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(792..794,795..797,798..1508)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(792..794,795..797,798..1508)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(837..858)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1098..1117)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1445..1466
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3158..3175)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3164..3388
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3521..3540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3672..3719
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3727..4749
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3727..4749
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4879..5000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4916..4935)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4970..4989
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5049..5065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5049..5065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5062..5084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5073..5089
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5097..5103,5104..5121,5122..5127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5142..5163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5280..5297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5451..6039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5531..5550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6210..7001,7002..7070))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6833..6852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7071..7175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7250..7269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctta tggtacctac gtatccttac gatgtgccag
      781 attacgccgg catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      841 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      901 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      961 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1021 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1081 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1141 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1201 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1261 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1321 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1381 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1441 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1501 tgtacaaggt cgacgccggc atggcagcca agaggagctc cggggccgcg ccagcacctg
     1561 cctcggcctc ggccccggcg ccggtgccgg gcggcgaggc cgagcgcgtc cgagtcttcc
     1621 acaaacaggc cttcgagtac atctccattg ccctgcgcat cgatgaggat gagaaagcag
     1681 gacagaagga gcaagctgtg gaatggtata agaaaggtat tgaagaactg gaaaaaggaa
     1741 tagctgttat agttacagga caaggtgaac agtgtgaaag agctagacgc cttcaagcta
     1801 aaatgatgac taatttggtt atggccaagg accgcttaca acttctagag aagatgcaac
     1861 cagttttgcc attttccaag tcacaaacgg acgtctataa tgacagtact aacttggcat
     1921 gccgcaatgg acatctccag tcagaaagtg gagctgttcc aaaaagaaaa gaccccttaa
     1981 cacacactag taattcactg cctcgttcaa aaacagtcat gaaaactgga tctgcaggcc
     2041 tttcaggcca ccatagagca cctagttaca gtggtttatc catggtttct ggagtgaaac
     2101 agggatctgg tcctgctcct accactcata agggtactcc gaaaacaaat aggacaaata
     2161 aaccttctac ccctacaact gctactcgta agaaaaaaga cttgaagaat tttaggaatg
     2221 tggacagcaa ccttgctaac cttataatga atgaaattgt ggacaatgga acagctgtta
     2281 aatttgatga tatagctggt caagacttgg caaaacaagc attgcaagaa attgttattc
     2341 ttccttctct gaggcctgag ttgttcacag ggcttagagc tcctgccaga gggctgttac
     2401 tctttggtcc acctgggaat gggaagacaa tgctggctaa agcagtagct gcagaatcga
     2461 atgcaacctt ctttaatata agtgctgcaa gtttaacttc aaaatacgtg ggagaaggag
     2521 agaaattggt gagggctctt tttgctgtgg ctcgagaact tcaaccttct ataattttta
     2581 tagatgaagt tgatagcctt ttgtgtgaaa gaagagaagg ggagcacgat gctagtagac
     2641 gcctaaaaac tgaatttcta atagaatttg atggtgtaca gtctgctgga gatgacagag
     2701 tacttgtaat gggtgcaact aataggccac aagagcttga tgaggctgtt ctcaggcgtt
     2761 ttatcaaacg ggtatatgtg tctttaccaa atgaggagac aagactactt ttgcttaaaa
     2821 atctgttatg taaacaagga agtccattga cccaaaaaga actagcacaa cttgctagaa
     2881 tgactgatgg atactcagga agtgacctaa cagctttggc aaaagatgca gcactgggtc
     2941 ctatccgaga actaaaacca gaacaggtga agaatatgtc tgccagtgag atgagaaata
     3001 ttcgattatc tgacttcact gaatccttga aaaaaataaa acgcagcgtc agccctcaaa
     3061 ctttagaagc gtacatacgt tggaacaagg actttggaga taccactgtt taagcggccg
     3121 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3181 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3241 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3301 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3361 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3421 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3481 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3541 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3601 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3661 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     3721 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     3781 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     3841 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     3901 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     3961 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     4021 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     4081 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     4141 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     4201 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     4261 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     4321 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     4381 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     4441 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     4501 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     4561 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     4621 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     4681 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     4741 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4801 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4861 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4921 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4981 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5041 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5101 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5161 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5281 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5341 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5401 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5461 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5521 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5581 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5641 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5701 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5761 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5821 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5881 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5941 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6001 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6061 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6121 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6181 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6241 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6301 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6361 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6421 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6481 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6541 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6601 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6661 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6721 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6781 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6841 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6901 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6961 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7021 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7081 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7141 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7201 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     7261 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7321 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     7381 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
//
