LOCUS       sequence-468557-        6826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose inducible expression of 3xFLAG-PolyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6826)
  AUTHORS   Chavez Lab
  TITLE     Multiplex screening identifies broad modifiers of ALS-associated 
            proteotoxicity
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6826)
  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            "unpublished"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-PolyA
FEATURES             Location/Qualifiers
     source          1..6826
                     /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(1928..1952)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1985..2001)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1986..2002)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2004..2251
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2020..2038)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2269..2288)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2270..2288)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2298..2315)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2298..2314)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2307..2329)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2455..2910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2542..2561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2752..2773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3041..3844)
                     /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(3845..4060)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4084..4103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4203..4225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4263..4281)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4322..5664
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4654..4701
                     /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        5691..5795
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5796..5864,5865..6656)
                     /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(6014..6033)
                     /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 agcttgcggc cgcgaattca gcagcagcag cagcagcagc agcagcagca
     1681 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca
     1741 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca
     1801 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca
     1861 gcagcagcag cagcagcagc agcagcagca gcagcagcag atctaacctc ctcagcttcc
     1921 tcatagaacc cagctttctt gtacaaagtg gtgatgggct gcaggaattc gatatcaagc
     1981 ttatcgatac cgtcgacctc gagtcatgta attagttatg tcacgcttac attcacgccc
     2041 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     2101 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     2161 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     2221 ggttttggga cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag
     2281 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2341 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2401 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     2461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2521 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2641 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2761 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2821 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2881 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     2941 gtgcggtatt tcacaccgca tagggtaata actgatataa ttaaattgaa gctctaattt
     3001 gtgagtttag tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc
     3061 ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc
     3121 ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat
     3181 catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg
     3241 gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa
     3301 agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc
     3361 cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct
     3421 ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt
     3481 gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt
     3541 tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg
     3601 cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca
     3661 catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct
     3721 tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa
     3781 atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg
     3841 acatgattta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg
     3901 ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct
     3961 ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc
     4021 gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa aaggtggtat ggtgcactct
     4081 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4141 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4201 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4261 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagta
     4321 tgatccaata tcaaaggaaa tgatagcatt gaaggatgag actaatccaa ttgaggagtg
     4381 gcagcatata gaacagctaa agggtagtgc tgaaggaagc atacgatacc ccgcatggaa
     4441 tgggataata tcacaggagg tactagacta cctttcatcc tacataaata gacgcatata
     4501 agtacgcatt taagcataaa cacgcactat gccgttcttc tcatgtatat atatatacag
     4561 gcaacacgca gatataggtg cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc
     4621 attttcggaa gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa gttcctattc
     4681 tctagaaagt ataggaactt cagagcgctt ttgaaaacca aaagcgctct gaagacgcac
     4741 tttcaaaaaa ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc
     4801 ttccacaaac attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata
     4861 taacctaccc atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt
     4921 ttatgtttat ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag
     4981 agtgaatcga aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa
     5041 atagaagaaa ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct
     5101 gttcacaaag tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt
     5161 gaaaaaatgc acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt
     5221 tttttatgga agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca
     5281 gtgcaaaaag ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct
     5341 aagatgcttt gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag
     5401 tatagattct ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc
     5461 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa
     5521 tgaagcacag attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa
     5581 aatgcagctc agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac
     5641 aaaatgaagc acagatgctt cgttcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5701 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5761 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5821 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5881 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5941 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6001 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6061 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6121 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6181 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6241 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6301 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6361 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6421 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6481 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6541 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6601 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6661 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6721 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6781 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
