LOCUS Exported 7019 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for the expression of the construct alpha-Synuclein-GFP, which contains N-terminal tags, which can be cleaved later: His-tag for purification and SUMO-tag for better solubility. ACCESSION . VERSION . KEYWORDS 1a. pETM11-SUMO-SNCA-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7019) AUTHORS Afitska K, Fucikova A, Shvadchak VV, Yushchenko DA TITLE Modification of C Terminus Provides New Insights into the Mechanism of alpha-Synuclein Aggregation. JOURNAL Biophys J. 2017 Nov 21;113(10):2182-2191. doi: 10.1016/j.bpj.2017.08.027. Epub 2017 Sep 20. PUBMED 28939194 REFERENCE 2 (bases 1 to 7019) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7019 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 24..43 /label=T7 /note="T7 promoter, forward primer" promoter 24..42 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 43..67 /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)." RBS 82..104 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 117..134 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 165..185 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 891..1604 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(933..954) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1194..1213) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1541..1562 /label=EGFP-C /note="EGFP, forward primer" CDS 1907..1924 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1977..1995) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1991..2038 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2075..2530 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2162..2181) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2372..2393 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2622..3437) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 3345..3364 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3429..3448 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3559..4147 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4048..4067 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4301..4318 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4333..4472 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4398..4417) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4558..4580 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4574..4765) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5574..6656) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 6618..6637 /label=LacI-R /note="LacI, reverse primer" promoter complement(6657..6734) /gene="lacI" /label=lacI promoter primer_bind 6940..6959 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa 61 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgaaacatc 121 accatcacca tcaccccatg agcgattacg acatccccac tactgagaat ctttattttc 181 agggcgccat gggcaacgat cacattaacc tgaaagtggc cggtcaagac ggtagcgtag 241 tccagtttaa aatcaaacgc cacacccctc tgtcgaaact gatgaaagcc tattgtgaac 301 gccaaggtct gtctatgcgt cagatccgtt ttcgcttcga tggacagccg attaacgaaa 361 ccgacactcc agcacagctg gaaatggaag atgaggacac cattgacgtg ttccagcaac 421 agaccggtgg atccatggat gtattcatga aaggactttc aaaggccaag gagggagttg 481 tggctgctgc tgagaaaacc aaacagggtg tggcagaagc agcaggaaag acaaaagagg 541 gtgttctcta tgtaggctcc aaaaccaagg agggagtggt gcatggtgtg gcaacagtgg 601 ctgagaagac caaagagcaa gtgacaaatg ttggaggagc agtggtgacg ggtgtgacag 661 cagtagccca gaagacagtg gagggagcag ggagcattgc agcagccact ggctttgtca 721 aaaaggacca gttgggcaag aatgaagaag gagccccaca ggaaggaatt ctggaagata 781 tgcctgtgga tcctgacaat gaggcttatg aaatgccttc tgaggaaggg tatcaagact 841 acgaacctga agcccatatg gctagcggcg gctctggtgg cggctctggt gtgagcaagg 901 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 961 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 1021 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 1081 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 1141 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 1201 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1261 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1321 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1381 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1441 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1501 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1561 tgaccgccgc cgggatcact ctcggcatgg acgagctcta caagtgaaag cttcggaaga 1621 agagccaggg cagcaagaat cctcagaagt actcggacaa ggagctgcag tacatcgacg 1681 ccatcagcaa caagcagggc gagctggaga attacgtgtc cgacggctac aagaccgcac 1741 tgacagagga gcgcaggcgc ttctgcttcc tggtggagaa gcagtgcgcc gtggccaaga 1801 actccgcggc ctaccactcc aagggcaagg agctgctggc gcagaagctg ccgctgtggc 1861 aacaggcctg tgccgacccc agcaagatct aagcggccgc ctcgagcacc accaccacca 1921 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc 1981 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct 2041 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc 2101 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 2161 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 2221 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 2281 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 2341 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 2401 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 2461 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 2521 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 2581 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc 2641 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 2701 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 2761 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 2821 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 2881 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 2941 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 3001 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 3061 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 3121 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 3181 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 3241 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 3301 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 3361 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 3421 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 3481 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 3541 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 3601 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 3661 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 3721 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 3781 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 3841 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 3901 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 3961 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 4021 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 4081 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 4141 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 4201 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 4261 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 4321 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 4381 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 4441 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 4501 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 4561 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt 4621 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca 4681 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa 4741 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg 4801 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac 4861 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc 4921 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac 4981 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact 5041 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg 5101 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct 5161 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca 5221 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg 5281 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg 5341 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg 5401 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga 5461 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 5521 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 5581 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 5641 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc 5701 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 5761 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 5821 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac 5881 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 5941 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 6001 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 6061 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 6121 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 6181 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 6241 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 6301 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 6361 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 6421 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 6481 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 6541 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 6601 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 6661 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 6721 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca 6781 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 6841 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc 6901 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc 6961 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagagga //