LOCUS       sequence-468556-        6829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose inducible expression of 3xFLAG-PolyL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6829)
  AUTHORS   Chavez Lab
  TITLE     Multiplex screening identifies broad modifiers of ALS-associated 
            proteotoxicity
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6829)
  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            "unpublished"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-3xFLAG-PolyL
FEATURES             Location/Qualifiers
     source          1..6829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1054..1495
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    1054..1171
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1466..1489
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1510..1526
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1510..1526
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1534..1558
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1567..1632
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(1931..1955)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1988..2004)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1989..2005)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2007..2254
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2023..2041)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2272..2291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2273..2291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2301..2318)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2301..2317)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2310..2332)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2458..2913
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2545..2564)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2755..2776
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3044..3847)
                     /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"
     promoter        complement(3848..4063)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4087..4106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4206..4228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4266..4284)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4325..5667
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4657..4704
                     /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)."
     promoter        5694..5798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5799..5867,5868..6659)
                     /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     complement(6017..6036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctcta gtacggatta gaagccgccg agcgggtgac
     1081 agccctccga aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa
     1141 acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt
     1201 tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga
     1261 atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta
     1321 attaatcagc gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca
     1381 taaccacttt aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta
     1441 ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa
     1501 accccggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa agcaggctac
     1561 aaaatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
     1621 gacgatgaca agcttcttct ccttctcctt ctccttctcc ttctccttct ccttctcctt
     1681 ctccttctcc ttctccttct ccttctcctt ctccttctcc ttctccttct ccttctcctt
     1741 ctccttctcc ttctccttct ccttctcctt ctccttctcc ttctccttct ccttctcctt
     1801 ctccttctcc ttctccttct ccttctcctt ctccttctcc ttctccttct ccttctcctt
     1861 ctccttctcc ttctccttct ccttctcctt ctccttcttc tagaaggact tgagggactc
     1921 gaaggctaga acccagcttt cttgtacaaa gtggtgatgg gctgcaggaa ttcgatatca
     1981 agcttatcga taccgtcgac ctcgagtcat gtaattagtt atgtcacgct tacattcacg
     2041 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     2101 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     2161 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     2221 gaaggttttg ggacgctcga aggctttaat ttgcggccgg tacccaattc gccctatagt
     2281 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2341 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2401 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg
     2461 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2521 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2581 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2641 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2701 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2761 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2821 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2881 cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcatagggta ataactgata taattaaatt gaagctctaa
     3001 tttgtgagtt tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc
     3061 atcttctcaa atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat
     3121 cccttccctt tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca
     3181 catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac
     3241 cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa
     3301 taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt
     3361 ctccagtaga tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt
     3421 cctttgttac ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac
     3481 cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca
     3541 atttgactgt attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact
     3601 tggcggataa tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat
     3661 ccacatgtgt ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt
     3721 ccttggtggt acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat
     3781 taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt
     3841 tcgacatgat ttatcttcgt ttcctgcagg tttttgttct gtgcagttgg gttaagaata
     3901 ctgggcaatt tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt
     3961 gctccttcct tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa
     4021 accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt gaaaaggtgg tatggtgcac
     4081 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4261 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4321 gtatgatcca atatcaaagg aaatgatagc attgaaggat gagactaatc caattgagga
     4381 gtggcagcat atagaacagc taaagggtag tgctgaagga agcatacgat accccgcatg
     4441 gaatgggata atatcacagg aggtactaga ctacctttca tcctacataa atagacgcat
     4501 ataagtacgc atttaagcat aaacacgcac tatgccgttc ttctcatgta tatatatata
     4561 caggcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt
     4621 tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta
     4681 ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg
     4741 cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac
     4801 cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct
     4861 atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat
     4921 tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca
     4981 tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac
     5041 aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt
     5101 tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat
     5161 cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg
     5221 ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct
     5281 acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa
     5341 tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag
     5401 aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa
     5461 tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa
     5521 aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt
     5581 aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc
     5641 tacaaaatga agcacagatg cttcgttcag gtggcacttt tcggggaaat gtgcgcggaa
     5701 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5761 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5821 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5881 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5941 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6001 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6061 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6121 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6181 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6241 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6301 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6361 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6421 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6481 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6541 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6601 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6661 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6721 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6781 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
