LOCUS       sequence-175017-        6915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETDUET-1 based vector for BiMolecular Fluorescence Complementation 
            based assays of alpha-synuclein oligomerisation .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  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 6915)
  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: pET-alpha-synuclein-BiFC
FEATURES             Location/Qualifiers
     source          1..6915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             427..678
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     615..636
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        790..808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    809..833
                     /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(1302..1304,1305..1307,1308..1766)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYLTA"
     primer_bind     complement(1347..1368)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1608..1627)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1794..1838
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1876..1894)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1890..1937
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1974..2429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2061..2080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2271..2292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2544..3335,3336..3404))
                     /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     3167..3186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3405..3497)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4320..4337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4352..4491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4417..4436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4577..4599
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4593..4784)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5356..6438)
                     /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     6400..6419
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6439..6516)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6722..6741
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6829..6847
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6848..6872
                     /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             6887..6909
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggatgtat tcatgaaagg actttcaaag gccaaggagg gagttgtggc tgctgctgag
       61 aaaaccaaac agggtgtggc agaagcagca ggaaagacaa aagagggtgt tctctatgta
      121 ggctccaaaa ccaaggaggg agtggtgcat ggtgtggcaa cagtggctga gaagaccaaa
      181 gagcaagtga caaatgttgg aggagcagtg gtgacgggtg tgacagcagt agcccagaag
      241 acagtggagg gagcagggag cattgcagca gccactggct ttgtcaaaaa ggaccagttg
      301 ggcaagaatg aagagggagc cccacaggaa ggaattctgg aagatatgcc tgtggatcct
      361 gacaatgagg cttatgaaat gccttctgag gaagggtatc aagactacga acctgaagcc
      421 ggatccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
      481 gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      541 gtgctgctgc ccgacaacca ctacctgagc taccagtcca aactgagcaa agaccccaac
      601 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      661 atggacgagc tgtacaagta aagatccgaa ttcgagctcg gcgcgcctgc aggtcgacaa
      721 gcttgcggcc gcataatgct taagtcgaac agaaagtaat cgtattgtac acggccgcat
      781 aatcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tccccatctt
      841 agtatattag ttaagtataa gaaggagata tacatatgga tgtattcatg aaaggacttt
      901 caaaggccaa ggagggagtt gtggctgctg ctgagaaaac caaacagggt gtggcagaag
      961 cagcaggaaa gacaaaagag ggtgttctct atgtaggctc caaaaccaag gagggagtgg
     1021 tgcatggtgt ggcaacagtg gctgagaaga ccaaagagca agtgacaaat gttggaggag
     1081 cagtggtgac gggtgtgaca gcagtagccc agaagacagt ggagggagca gggagcattg
     1141 cagcagccac tggctttgtc aaaaaggacc agttgggcaa gaatgaagag ggagccccac
     1201 aggaaggaat tctggaagat atgcctgtgg atcctgacaa tgaggcttat gaaatgcctt
     1261 ctgaggaagg gtatcaagac tacgaacctg aagccagatc tatggtgagc aagggcgagg
     1321 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1381 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagc
     1441 tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgggct
     1501 acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1561 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1621 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1681 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1741 acagccacaa cgtctatctc accgcctgag gatccggtac cctcgagtct ggtaaagaaa
     1801 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     1861 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1921 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1981 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2041 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2101 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2161 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2221 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2281 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2341 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2401 ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag attatcaaaa
     2461 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2521 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2581 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2641 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2701 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2761 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2821 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2881 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2941 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3001 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3061 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3121 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3181 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3241 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3301 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3361 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3421 tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3481 ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4261 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4321 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4381 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4441 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4501 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4561 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4621 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4681 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4741 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4801 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4861 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4921 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4981 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5041 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5101 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5161 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5221 aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga gctgactggg
     5281 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5341 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5401 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5461 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5521 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5581 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     5641 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5701 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5761 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5821 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5881 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5941 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     6001 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     6061 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6121 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6181 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6241 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6301 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6361 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6421 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6481 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6541 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6601 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6661 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6721 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6781 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca
     6841 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     6901 agaaggagat atacc
//
