LOCUS       sequence-468511-        6835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose inducible expression of 3xFLAG-PolyC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6835)
  AUTHORS   Chavez Lab
  TITLE     Multiplex screening identifies broad modifiers of ALS-associated 
            proteotoxicity
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6835)
  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            "unpublished"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-PolyC
FEATURES             Location/Qualifiers
     source          1..6835
                     /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(1937..1961)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1994..2010)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1995..2011)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2013..2260
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2029..2047)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2278..2297)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2279..2297)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2307..2324)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2307..2323)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2316..2338)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2464..2919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2551..2570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2761..2782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3050..3853)
                     /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(3854..4069)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4093..4112)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4212..4234
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4272..4290)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4331..5673
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4663..4710
                     /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        5700..5804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5805..5873,5874..6665)
                     /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(6023..6042)
                     /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 agctttgttg ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt
     1681 tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt
     1741 tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt
     1801 tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt
     1861 tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg gaaagatctg
     1921 agggactcga agtagaaccc agctttcttg tacaaagtgg tgatgggctg caggaattcg
     1981 atatcaagct tatcgatacc gtcgacctcg agtcatgtaa ttagttatgt cacgcttaca
     2041 ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
     2101 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
     2161 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
     2221 gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc
     2281 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     2341 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2401 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2461 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2521 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2581 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2641 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2701 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2761 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2821 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2881 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     2941 tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag
     3001 ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct
     3061 ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct
     3121 tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag
     3181 agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac
     3241 ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt
     3301 gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag
     3361 tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc
     3421 taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca
     3481 ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt
     3541 actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat
     3601 tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca
     3661 agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca
     3721 gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca
     3781 tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg
     3841 tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta
     3901 agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa
     3961 gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc
     4021 aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg
     4081 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4141 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4201 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4261 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4321 ttcttagtat gatccaatat caaaggaaat gatagcattg aaggatgaga ctaatccaat
     4381 tgaggagtgg cagcatatag aacagctaaa gggtagtgct gaaggaagca tacgataccc
     4441 cgcatggaat gggataatat cacaggaggt actagactac ctttcatcct acataaatag
     4501 acgcatataa gtacgcattt aagcataaac acgcactatg ccgttcttct catgtatata
     4561 tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
     4621 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
     4681 ttcctattct ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg
     4741 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
     4801 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
     4861 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
     4921 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
     4981 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
     5041 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
     5101 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
     5161 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
     5221 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
     5281 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
     5341 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
     5401 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
     5461 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     5521 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
     5581 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     5641 ttgttctaca aaatgaagca cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc
     5701 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5761 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5821 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5881 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5941 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6001 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6061 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6121 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6181 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6241 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6301 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6361 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6421 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6481 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6541 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6601 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6661 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6721 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6781 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
