LOCUS       sequence-211385-        6557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of human alpha-synuclein, carboxyl 
            terminally tagged with mCerulean3 and poly-histidines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6557)
  AUTHORS   Eastwood T, Baker K, Brooker H, Frank S, Mulvihill DP
  TITLE     An enhanced recombinant amino-terminal acetylation system and novel 
            in vivo high-throughput screen for molecules affecting 
            alpha-synuclein oligomerisation.
  JOURNAL   FEBS Lett. 2017 Feb 18. doi: 10.1002/1873-3468.12597.
  PUBMED    28214355
REFERENCE   2  (bases 1 to 6557)
  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: "FEBS Lett. 
            2017 Feb 18. doi: 10.1002/1873-3468.12597."
COMMENT     Sequence Label: pDUET-1-alpha-synuclein-mCerulean3-His6
FEATURES             Location/Qualifiers
     source          1..6557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             38..60
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             79..96
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        210..228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    229..253
                     /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)."
     CDS             join(731..733,734..736,737..1447)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(776..797)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1037..1056)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1384..1405
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1457..1474
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1502..1546
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1584..1602)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1598..1645
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1682..2137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1769..1788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1979..2000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2252..3043,3044..3112))
                     /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     2875..2894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3113..3205)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3286..3874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3775..3794
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4028..4045
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4060..4199
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4125..4144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4285..4307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4301..4492)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5064..6146)
                     /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     6108..6127
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6147..6224)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6430..6449
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6537..6555
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    join(6556..6557,1..23)
                     /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)."
ORIGIN
        1 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
       61 tataccatgg gcagcagcca tcaccatcat caccacagcc aggatccgaa ttcgagctcg
      121 gcgcgcctgc aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat
      181 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc
      241 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatgga
      301 tgtattcatg aaaggacttt caaaggccaa ggagggagtt gtggctgctg ctgagaaaac
      361 caaacagggt gtggcagaag cagcaggaaa gacaaaagag ggtgttctct atgtaggctc
      421 caaaaccaag gagggagtgg tgcatggtgt ggcaacagtg gctgagaaga ccaaagagca
      481 agtgacaaat gttggaggag cagtggtgac gggtgtgaca gcagtagccc agaagacagt
      541 ggagggagca gggagcattg cagcagccac tggctttgtc aaaaaggacc agttgggcaa
      601 gaatgaagag ggagccccac aggaaggaat tctggaagat atgcctgtgg atcctgacaa
      661 tgaggcttat gaaatgcctt ctgaggaagg gtatcaagac tacgaacctg aagccggatc
      721 cggaggagga atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      781 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      841 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      901 ctggcccacc ctcgtgacca ccctgagctg gggcgtgcag tgcttcgccc gctaccccga
      961 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1021 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1081 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1141 cctggggcac aagctggagt acaacgccat ccacggcaac gtctatatca ccgccgacaa
     1201 gcagaagaac ggcatcaagg ccaacttcgg gctcaactgc aacatcgagg acggcagcgt
     1261 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1321 cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga
     1381 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1441 gtacaaggga ggaggacatc atcatcatca tcattaaaga tctggtaccc tcgagtctgg
     1501 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc
     1561 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1621 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1681 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1741 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1801 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1861 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1921 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1981 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2041 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2101 aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat
     2161 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2221 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2281 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2341 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     2401 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2461 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2521 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2581 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2641 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2701 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2761 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2821 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2881 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2941 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3001 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3061 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3121 tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3181 gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc
     3241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3421 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3661 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3721 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3781 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3841 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3901 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3961 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4021 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4081 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4141 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4201 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4261 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4321 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4381 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4441 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4501 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4561 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4621 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4681 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4741 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4801 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4861 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4921 gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc
     4981 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5041 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5101 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5161 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5221 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5281 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5341 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5401 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5461 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5521 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5581 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5641 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5701 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5761 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5821 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5881 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5941 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     6001 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     6061 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6121 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     6181 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     6241 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6301 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6361 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6421 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6481 gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat
     6541 acgactcact atagggg
//
