LOCUS Exported 4264 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Inducible expression of M87 S245X spastin in mammalian cells. ACCESSION . VERSION . KEYWORDS pTRE/SP M87 S245X SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4264) AUTHORS Solowska JM, Rao AN, Baas PW TITLE Studies on truncating mutations of SPAST associated with Hereditary Spastic Paraplegia indicate greater accumulation and toxicity of the M1 isoform of spastin. JOURNAL Mol Biol Cell. 2017 May 11. pii: mbc.E17-01-0047. doi: 10.1091/mbc.E17-01-0047. PUBMED 28495799 REFERENCE 2 (bases 1 to 4264) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4264 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..315 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 9..27 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 45..63 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 80..98 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 116..134 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 152..170 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 187..205 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 223..241 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 288..312 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 350..359 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2175..2256 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2212..2231) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(2267..2284) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2438..3026) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2518..2537) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3197..4057) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3820..3839 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4058..4162) /gene="bla" /label=AmpR promoter primer_bind 4230..4248 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag 61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct 121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg 181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag 241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 301 aaccgtcaga tcgcctggag aattcgagct cggtacccgc ttctcccgcg ccaccatggc 361 agccaagagg agctccgggg ccgcgccagc acctgcctcg gcctcggccc cggcgccggt 421 gccgggcggc gaggccgagc gcgtccgagt cttccacaaa caggccttcg agtacatctc 481 cattgccctg cgcatcgatg aggatgagaa agcaggacag aaggagcaag ctgtggaatg 541 gtataagaaa ggtattgaag aactggaaaa aggaatagct gttatagtta caggacaagg 601 tgaacagtgt gaaagagcta gacgccttca agctaaaatg atgactaatt tggttatggc 661 caaggaccgc ttacaacttc tagagaagat gcaaccagtt ttgccatttt ccaagtcaca 721 aacggacgtc tataatgaca gtactaactt ggcatgccgc aatggacatc tccagtcaga 781 aagtggagct gttccaaaaa gaaaagaccc cttaacacac actagtaatt gactgcctcg 841 ttcaaaaaca gttatgaaaa ctggatctgc aggcctttca ggccaccata gagcacctag 901 ttacagtggt ttatccatgg tttctggagt gaaacaggga tctggtcctg ctcctaccac 961 tcataagggt actccgaaaa caaataggac aaataaacct tctaccccta caactgctac 1021 tcgtaagaaa aaagacttga agaattttag gaatgtggac agcaaccttg ctaaccttat 1081 aatgaatgaa attgtggaca atggaacagc tgttaaattt gatgatatag ctggtcaaga 1141 cttggcaaaa caagcattgc aagaaattgt tattcttcct tctctgaggc ctgagttgtt 1201 cacagggctt agagctcctg ccagagggct gttactcttt ggtccacctg ggaatgggaa 1261 gacaatgctg gctaaagcag tagctgcaga atcgaatgca accttcttta atataagtgc 1321 tgcaagttta acttcaaaat acgtgggaga aggagagaaa ttggtgaggg ctctttttgc 1381 tgtggctcga gaacttcaac cttctataat ttttatagat gaagttgata gccttttgtg 1441 tgaaagaaga gaaggggagc acgatgctag tagacgccta aaaactgaat ttctaataga 1501 atttgatggt gtacagtctg ctggagatga cagagtactt gtaatgggtg caactaatag 1561 gccacaagag cttgatgagg ctgttctcag gcgtttcatc aaacgggtat atgtgtcttt 1621 accaaatgag gagacaagac tacttttgct taaaaatctg ttatgtaaac aaggaagtcc 1681 attgacccaa aaagaactag cacaacttgc tagaatgact gatggatact caggaagtga 1741 cctaacagct ttggcaaaag atgcagcact gggtcctatc cgagaactaa aaccagaaca 1801 ggtgaagaat atgtctgcca gtgagatgag aaatattcga ttatctgact tcactgaatc 1861 cttgaaaaaa ataaaacgca gcgtcagccc tcaaacttta gaagcgtaca tacgttggaa 1921 caaggacttt ggagatacca ctgtttaagg aaataccttt gtaaacctgc agaacatttt 1981 acttaaaaga ggaaacacaa gatcctctag tcagctgacg cgtgctagcg cggccgcatc 2041 gataagcttg tcgacgatat ctctagagga tcataatcag ccataccaca tttgtagagg 2101 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 2161 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2221 tcacaaattt cacaaataaa gcattttttt cactgcctcg agcttcctcg ctcactgact 2281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 2341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 2401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 2461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 2521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 2581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 2641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 2701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 2761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 2821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 2881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 2941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 3121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 3181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 3241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 3301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 3361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 3421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 3481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 3541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 3601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 3661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 3721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 3781 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca 3841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 3901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 3961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 4021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 4081 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 4141 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 4201 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc 4261 actc //