LOCUS       sequence-329866-        3724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of alpha synuclein coupled to GFP via a TEV 
            site for uptake studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3724)
  AUTHORS   Jarvela TS, Chaplot K, Lindberg I
  TITLE     A protease protection assay for the detection of internalized 
            alpha-synuclein pre-formed fibrils.
  JOURNAL   PLoS One. 2021 Jan 26;16(1):e0241161. doi: 
            10.1371/journal.pone.0241161. eCollection 2021.
  PUBMED    33497415
REFERENCE   2  (bases 1 to 3724)
  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: "PLoS One.";
            date: "2021-01-26"; volume: "16(1):e0241161. doi"; pages: " 
            10.1371/journal.pone.0241161. eCollection 2021"
COMMENT     Sequence Label: pRK172-alpha-syn-TEV-GFP
FEATURES             Location/Qualifiers
     source          1..3724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             501..518
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             522..542
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(543..545,546..548,549..1262)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(543..545,546..548,549..1259)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(588..609)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(849..868)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1196..1217
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1323..1341)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1337..1384
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1429..1447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1515..1619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1620..1688,1689..2480)
                     /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(1838..1857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2651..3239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3140..3159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3393..3410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3425..3564
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3490..3509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3650..3672
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atggatgtgt tcatgaaagg actttcaaag gccaaggagg
      121 gagttgtggc tgctgctgag aaaaccaagc agggtgtggc agaggcagct ggaaagacaa
      181 aagagggagt cctctatgta ggttccaaaa ctaaggaagg agtggttcat ggagtgacaa
      241 cagtggctga gaagaccaaa gagcaagtga caaatgttgg aggagcagtg gtgactggtg
      301 tgacagcagt cgctcagaag acagtggagg gagctgggaa tatagctgct gccactggct
      361 ttgtcaagaa ggaccagatg ggcaagggtg aggaggggta cccacaggaa ggaatcctgg
      421 aagacatgcc tgtggatcct ggcagtgagg cttatgaaat gccttcagag gaaggctacc
      481 aagactatga gcctgaagcc caccatcacc atcaccatct cgagaatctt tattttcagg
      541 gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      601 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      661 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      721 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      781 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      841 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      901 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      961 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1021 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1081 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1141 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1201 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1261 aagcttgaat tccgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1321 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1381 tttgctgaaa ggaggaacta tatccggata attcttgaag acgaaagggc ctcgtgatac
     1441 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1561 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1621 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1681 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1741 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1801 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1861 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1921 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1981 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2041 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2101 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2161 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2221 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2281 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2341 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2401 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2461 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2521 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2761 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3301 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3361 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3421 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3481 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3541 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3601 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3661 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcaggatct cgatcccgcg
     3721 aaat
//
