LOCUS       sequence-332012-        7440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For doxycycline inducible mammalian expression of EGFP tagged 
            Spastin M87 with exon 4, with N386C mutation..
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, N386C
FEATURES             Location/Qualifiers
     source          1..7440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     255..279
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             382..408
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(412..414,415..417,418..1128)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(412..414,415..417,418..1128)
                     /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(457..478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(718..737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1065..1086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2778..2795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2784..3008
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3141..3160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3292..3339
                     /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             3347..4369
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3347..4369
                     /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    4499..4620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4536..4555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4590..4609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4669..4685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4669..4685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4682..4704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4693..4709
                     /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(4717..4723,4724..4741,4742..4747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4762..4783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4900..4917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5071..5659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5151..5170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5830..6621,6622..6690))
                     /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     6453..6472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6691..6795)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6870..6889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7061..7440
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      241 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      301 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      361 cttaagctta tggtacctac gtatccttac gatgtgccag attacgccgg catggtgagc
      421 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      481 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      541 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      601 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
      661 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
      721 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
      781 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
      841 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
      901 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
      961 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1021 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1081 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggt cgacgccggc
     1141 atggcagcca agaggagctc cggggccgcg ccagcacctg cctcggcctc ggccccggcg
     1201 ccggtgccgg gcggcgaggc cgagcgcgtc cgagtcttcc acaaacaggc cttcgagtac
     1261 atctccattg ccctgcgcat cgatgaggat gagaaagcag gacagaagga gcaagctgtg
     1321 gaatggtata agaaaggtat tgaagaactg gaaaaaggaa tagctgttat agttacagga
     1381 caaggtgaac agtgtgaaag agctagacgc cttcaagcta aaatgatgac taatttggtt
     1441 atggccaagg accgcttaca acttctagag aagatgcaac cagttttgcc attttccaag
     1501 tcacaaacgg acgtctataa tgacagtact aacttggcat gccgcaatgg acatctccag
     1561 tcagaaagtg gagctgttcc aaaaagaaaa gaccccttaa cacacactag taattcactg
     1621 cctcgttcaa aaacagtcat gaaaactgga tctgcaggcc tttcaggcca ccatagagca
     1681 cctagttaca gtggtttatc catggtttct ggagtgaaac agggatctgg tcctgctcct
     1741 accactcata agggtactcc gaaaacaaat aggacaaata aaccttctac ccctacaact
     1801 gctactcgta agaaaaaaga cttgaagaat tttaggaatg tggacagcaa ccttgctaac
     1861 cttataatga atgaaattgt ggacaatgga acagctgtta aatttgatga tatagctggt
     1921 caagacttgg caaaacaagc attgcaagaa attgttattc ttccttctct gaggcctgag
     1981 ttgttcacag ggcttagagc tcctgccaga gggctgttac tctttggtcc acctgggtgt
     2041 gggaagacaa tgctggctaa agcagtagct gcagaatcga atgcaacctt ctttaatata
     2101 agtgctgcaa gtttaacttc aaaatacgtg ggagaaggag agaaattggt gagggctctt
     2161 tttgctgtgg ctcgagaact tcaaccttct ataattttta tagatgaagt tgatagcctt
     2221 ttgtgtgaaa gaagagaagg ggagcacgat gctagtagac gcctaaaaac tgaatttcta
     2281 atagaatttg atggtgtaca gtctgctgga gatgacagag tacttgtaat gggtgcaact
     2341 aataggccac aagagcttga tgaggctgtt ctcaggcgtt ttatcaaacg ggtatatgtg
     2401 tctttaccaa atgaggagac aagactactt ttgcttaaaa atctgttatg taaacaagga
     2461 agtccattga cccaaaaaga actagcacaa cttgctagaa tgactgatgg atactcagga
     2521 agtgacctaa cagctttggc aaaagatgca gcactgggtc ctatccgaga actaaaacca
     2581 gaacaggtga agaatatgtc tgccagtgag atgagaaata ttcgattatc tgacttcact
     2641 gaatccttga aaaaaataaa acgcagcgtc agccctcaaa ctttagaagc gtacatacgt
     2701 tggaacaagg actttggaga taccactgtt taagcggccg ctcgagtcta gagggcccgt
     2761 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2821 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2881 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2941 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3001 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3061 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3121 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3181 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3241 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     3301 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     3361 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3421 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3481 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     3541 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     3601 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     3661 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     3721 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     3781 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     3841 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     3901 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     3961 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4021 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4081 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4141 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4201 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4261 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4321 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     4381 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4441 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4501 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4561 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4621 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4681 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4741 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4801 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4861 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4921 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4981 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5041 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5101 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5161 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5221 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5281 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5341 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5401 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5461 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5521 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5581 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5641 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5701 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5761 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5821 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5881 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5941 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6001 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6061 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6121 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6181 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6241 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6301 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6361 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6421 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6481 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6541 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6601 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6661 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6721 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6781 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6841 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6901 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     6961 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     7021 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     7081 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     7141 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     7201 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     7261 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     7321 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     7381 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
//
