LOCUS       sequence-365324-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of M87 Spastin (A2C mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7058)
  AUTHORS   Cada AK, Pavlin MR, Castillo JP, Tong AB, Larsen KP, Ren X, Yokom 
            AL, Tsai FC, Shiah JV, Bassereau PM, Bustamante CJ, Hurley JH
  TITLE     Friction-driven membrane scission by the human ESCRT-III proteins 
            CHMP1B and IST1.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Jul 19;119(29):e2204536119. doi: 
            10.1073/pnas.2204536119. Epub 2022 Jul 11.
  PUBMED    35858336
REFERENCE   2  (bases 1 to 7058)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-07-19"; volume: "119(29):e2204536119. 
            doi"; pages: " 10.1073/pnas.2204536119. Epub 2022 Jul 11"
COMMENT     Sequence Label: His-GST-Cys-M87 Spastin
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     137..154
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    169..308
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(234..253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     394..416
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(410..601)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2059..2078
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2162..2181
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2162..2180
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2269..2291
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2311..2328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2338..2991
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             3001..3021
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4749..4767)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4763..4810
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5176..5181,5182..5198,5199..5204))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5231..5249)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5317..5421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5422..5490,5491..6282)
                     /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     complement(5640..5659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6453..7041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6942..6961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
       61 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
      121 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
      181 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
      241 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
      301 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
      361 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
      421 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
      481 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
      541 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
      601 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
      661 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
      721 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
      781 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
      841 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
      901 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
      961 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     1021 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     1081 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     1141 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     1201 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     1261 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     1321 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     1381 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     1441 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     1501 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     1561 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     1621 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     1681 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     1741 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     1801 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     1861 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     1921 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     1981 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     2041 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     2101 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     2161 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
     2221 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
     2281 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatgg ttcttctatg
     2341 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     2401 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     2461 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     2521 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     2581 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     2641 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     2701 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     2761 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     2821 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     2881 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     2941 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa agggatcgag
     3001 gaaaacctgt acttccaatc caatgcaatg tgcgcagcaa aacgtagcag cggtgcagca
     3061 ccggcaccgg caagtgctag tgcccctgca ccggttccag gtggtgaagc agaacgtgtt
     3121 cgtgtttttc ataaacaggc ctttgagtat attagcattg ccctgcgtat tgatgaagat
     3181 gaaaaagcag gtcagaaaga acaggcagtt gagtggtaca aaaaaggcat tgaagaactg
     3241 gaaaaaggta ttgccgttat tgttaccggt cagggtgaac agtgtgaacg tgcccgtcgt
     3301 ctgcaggcaa aaatgatgac caatctggtt atggcaaaag atcgcctgca gctgctggaa
     3361 aaaatgcagc cggttctgcc gtttagcaaa agccagaccg atgtttataa tgatagcaca
     3421 aatctggcct gccgtaatgg tcatctgcag agcgaaagcg gtgccgttcc gaaacgtaaa
     3481 gatccgctga cacataccag caatagcctg cctcgtagca aaaccgttat gaaaaccggt
     3541 agcgcaggtc tgagcggtca tcatcgtgca ccgagctata gcggtctgag catggttagc
     3601 ggtgttaaac aaggtagcgg tccggcaccg accacacata aaggcacccc gaaaaccaat
     3661 cgtaccaata aaccgagcac gccgaccacc gcaacacgta aaaagaaaga tctgaaaaac
     3721 ttccgcaacg tggatagcaa tctggcaaat ctgattatga acgagattgt ggataatggc
     3781 accgcagtga aatttgatga tattgcaggt caggatctgg ccaaacaggc actgcaagaa
     3841 attgttattc tgccgagcct gcgtcctgaa ctgtttaccg gtctgcgtgc tccggcacgt
     3901 ggtctgctgc tgtttggtcc gcctggtaat ggtaaaacca tgctggcaaa agcagttgca
     3961 gcagaaagca atgcaacctt ttttaacatt agcgcagcaa gcctgaccag caaatatgtt
     4021 ggtgaaggtg aaaaactggt tcgtgcactg tttgcagttg cccgtgaact gcagccgagc
     4081 attattttca ttgatgaggt tgatagcctg ctgtgcgaac gtcgtgaagg cgaacatgat
     4141 gcaagtcgtc gtctgaaaac cgaatttctg attgaatttg atggtgttca gagtgccggt
     4201 gatgatcgtg ttctggtgat gggtgccacc aatcgtccgc aagagctgga tgaagcagtt
     4261 ctgcgtcgtt ttatcaaacg tgtttatgtt agcctgccga atgaagaaac acgtctgctg
     4321 ttactgaaaa atctgctgtg taaacagggt agtccgctga cgcaaaaaga actggcacag
     4381 ctggcacgta tgaccgatgg ttattcaggt agcgatctga ccgcactggc caaagatgca
     4441 gcactgggtc cgattcgtga actgaaaccg gaacaggtta aaaacatgag cgcaagcgaa
     4501 atgcgtaaca ttcgtctgag cgattttacc gaaagcctga aaaaaatcaa acgtagcgtt
     4561 agtccgcaga cactggaagc atatattcgt tggaataaag attttggcga taccaccgtt
     4621 taataacatt ggaagtggat aacggatccg cgatcgcggc gcgccacctg gtggccggcc
     4681 ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     4741 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4801 gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt gtggtcgcca
     4861 tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg cggcggccaa
     4921 agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg
     4981 ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc
     5041 ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga
     5101 tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact
     5161 gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga
     5221 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5281 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5341 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5401 taacattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5461 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5521 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5581 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5641 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     5701 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5761 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5821 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5881 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5941 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     6001 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6061 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6121 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6181 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6241 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6301 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6361 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6421 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6481 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6541 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6601 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6661 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6721 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6781 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6841 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6901 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6961 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7021 ggggggcgga gcctatggaa aaacgccagc aacgcggc
//
