LOCUS       sequence-439543-        6886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose inducible expression of Alpha-synuclein-E46K.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6886)
  AUTHORS   Chavez Lab
  TITLE     Multiplex screening identifies broad modifiers of ALS-associated 
            proteotoxicity
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6886)
  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: 
            "unpublished"
COMMENT     Sequence Label: pAG426-GAL-Alpha-synuclein-E46K
FEATURES             Location/Qualifiers
     source          1..6886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     183..201
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     219..235
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     220..236
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    269..293
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(723..747)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(755..771)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(755..771)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(786..1227)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(792..815)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1110..1227
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(1251..1271)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1251..1269)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1290..1306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1290..1306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1303..1325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1314..1330
                     /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)."
     promoter        complement(join(1338..1344,1345..1362,1363..1368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1383..1404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1521..1538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1692..2280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1772..1791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2451..3242,3243..3311))
                     /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     3074..3093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3312..3416)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3443..4785
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4406..4453)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     4826..4844
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4882..4904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5004..5023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5047..5262
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             5263..6066
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(6197..6652)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6334..6355)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6546..6565
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6778..6800
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6792..6809
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6793..6809
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6819..6838
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6819..6837
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(join(6856..6886,1..217))
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa
       61 agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata
      121 gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga
      181 gggcgtgaat gtaagcgtga cataactaat tacatgactc gaggtcgacg gtatcgataa
      241 gcttgatatc gaattcctgc agcccatcac cactttgtac aagaaagctg ggttttaggc
      301 ttcaggttcg tagtcttgat acccttcctc agaaggcatt tcataagcct cattgtcagg
      361 atccacaggc atatcttcca gaattccttc ctgtggggct ccttcttcat tcttgcccaa
      421 ctggtccttt ttgacaaagc cagtggctgc tgcaatgctc cctgctccct ccactgtctt
      481 ctgggctact gctgtcacac ccgtcaccac tgctcctcca acatttgtca cttgctcttt
      541 ggtcttctca gccactgttg ccacaccatg caccactccc ttcttggttt tggagcctac
      601 atagagaaca ccctcttttg tctttcctgc tgcttctgcc acaccctgtt tggttttctc
      661 agcagcagcc acaactccct ccttggcctt tgaaagtcct ttcatgaata catccatttt
      721 gtagcctgct tttttgtaca aacttgtgat gggggatcca ctagttctag aatccggggt
      781 tttttctcct tgacgttaaa gtatagaggt atattaacaa ttttttgttg atacttttat
      841 tacatttgaa taagaagtaa tacaaaccga aaatgttgaa agtattagtt aaagtggtta
      901 tgcagttttt gcatttatat atctgttaat agatcaaaaa tcatcgcttc gctgattaat
      961 taccccagaa ataaggctaa aaaactaatc gcattatcat cctatggttg ttaatttgat
     1021 tcgttcattt gaaggtttgt ggggccaggt tactgccaat ttttcctctt cataaccata
     1081 aaagctagta ttgtagaatc tttattgttc ggagcagtgc ggcgcgaggc acatctgcgt
     1141 ttcaggaacg cgaccggtga agacgaggac gcacggagga gagtcttcct tcggagggct
     1201 gtcacccgct cggcggcttc taatccgtac tagagctcca gcttttgttc cctttagtga
     1261 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1321 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1381 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1501 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1561 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1621 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1681 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1741 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1801 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1861 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1921 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1981 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2041 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2101 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2161 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2221 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2281 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2341 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2401 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2461 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2521 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2581 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2641 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2701 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2761 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2821 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2881 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2941 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3001 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3061 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3121 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3181 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3241 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3301 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3421 tttccccgaa aagtgccacc tgaacgaagc atctgtgctt cattttgtag aacaaaaatg
     3481 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa
     3541 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa
     3601 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     3661 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc
     3721 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt
     3781 ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa
     3841 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac
     3901 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc
     3961 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg
     4021 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata
     4081 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct
     4141 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc
     4201 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc
     4261 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat
     4321 attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag
     4381 cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg
     4441 gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc
     4501 gcacatacag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat
     4561 acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt
     4621 tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta
     4681 tcgtatgctt ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg
     4741 attagtctca tccttcaatg ctatcatttc ctttgatatt ggatcatact aagaaaccat
     4801 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     4861 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     4921 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     4981 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatac
     5041 caccttttca attcatcatt ttttttttat tctttttttt gatttcggtt tccttgaaat
     5101 ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga
     5161 ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc
     5221 caactgcaca gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat
     5281 aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac
     5341 gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag
     5401 ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact
     5461 gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt
     5521 ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct
     5581 gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc
     5641 ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc
     5701 cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag
     5761 ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga
     5821 gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta
     5881 gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca
     5941 ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta
     6001 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa
     6061 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca
     6121 atttaattat atcagttatt accctatgcg gtgtgaaata ccgcacagat gcgtaaggag
     6181 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     6241 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     6301 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     6361 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     6421 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     6481 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     6541 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     6601 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
     6661 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     6721 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     6781 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
     6841 aattgggtac cggccgcaaa ttaaagcctt cgagcgtccc aaaacc
//
