LOCUS       sequence-360304-        9289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible Shigella flexneri IpgD for expression in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9289)
  AUTHORS   Walpole GFW, Pacheco J, Chauhan N, Clark J, Anderson KE, Abbas YM, 
            Brabant-Kirwan D, Montano-Rendon F, Liu Z, Zhu H, Brumell JH, 
            Deiters A, Stephens LR, Hawkins PT, Hammond GRV, Grinstein S, Fairn 
            GD
  TITLE     Kinase-independent synthesis of 3-phosphorylated phosphoinositides 
            by a phosphotransferase.
  JOURNAL   Nat Cell Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y.
  PUBMED    35484249
REFERENCE   2  (bases 1 to 9289)
  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: "Nat Cell 
            Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y."
COMMENT     Sequence Label: pESC-LEU-IpgD
FEATURES             Location/Qualifiers
     source          1..9289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..42
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(80..102)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     202..221
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(715..1809)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1810..2217)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2518..2973)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2655..2676)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2867..2886
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3052..3217)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3138..3162
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3365..3388)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        3435..4099
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3651..3768
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4063..4086
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     terminator      5747..5936
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6008..6025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6179..6767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6259..6278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6938..7729,7730..7798))
                     /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     7561..7580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7799..7903)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7930..9272
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(8893..8940)
                     /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)."
ORIGIN
        1 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
       61 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
      121 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
      181 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatcg
      241 actacgtcgt taaggccgtt tctgacagag taaaattctt gagggaactt tcaccattat
      301 gggaaatggt tcaagaaggt attgacttaa actccatcaa atggtcaggt cattgagtgt
      361 tttttatttg ttgtattttt ttttttttag agaaaatcct ccaatatata aattaggaat
      421 catagtttca tgattttctg ttacacctaa ctttttgtgt ggtgccctcc tccttgtcaa
      481 tattaatgtt aaagtgcaat tctttttcct tatcacgttg agccattagt atcaatttgc
      541 ttacctgtat tcctttacat cctccttttt ctccttcttg ataaatgtat gtagattgcg
      601 tatatagttt cgtctaccct atgaacatat tccattttgt aatttcgtgt cgtttctatt
      661 atgaatttca tttataaagt ttatgtacaa atatcataaa aaaagagaat ctttttaagc
      721 aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac tgttggaacc
      781 acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat cttcaatggc
      841 cttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag acaagatagt
      901 ggcgataggg ttgaccttat tctttggcaa atctggagca gaaccgtggc atggttcgta
      961 caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg gcaacaaacc
     1021 caaggaacct gggataacgg aggcttcatc ggagatgata tcaccaaaca tgttgctggt
     1081 gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg cagaatcaat
     1141 caattgatgt tgaaccttca atgtagggaa ttcgttcttg atggtttcct ccacagtttt
     1201 tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa taggcaatgg
     1261 tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca cttctggaac
     1321 ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc tcttaccaaa
     1381 gtaaatacct cccactaatt ctctgacaac aacgaagtca gtacctttag caaattgtgg
     1441 cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc ttaagttggc
     1501 gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa cactaccggt
     1561 accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct tggaggcttc
     1621 cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac caattaaatg
     1681 attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa gaaccttaat
     1741 ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga tcttcttagg
     1801 ggcagacatt agaatggtat atccttgaaa tatatatata tatattgctg aaatgtaaaa
     1861 ggtaagaaaa gttagaaagt aagacgattg ctaaccacct attggaaaaa acaataggtc
     1921 cttaaataat attgtcaact tcaagtattg tgatgcaagc atttagtcat gaacgcttct
     1981 ctattctata tgaaaagccg gttccggcgc tctcaccttt cctttttctc ccaatttttc
     2041 agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc
     2101 gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa
     2161 tggttgctaa gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttaac
     2221 tgcggtcaag atatttcttg aatcaggcgc cttagaccgc tcggccaaac aaccaattac
     2281 ttgttgagaa atagagtata attatcctat aaatataacg tttttgaaca cacatgaaca
     2341 aggaagtaca ggacaattga ttttgaagag aatgtggatt ttgatgtaat tgttgggatt
     2401 ccatttttaa taaggcaata atattaggta tgtagatata ctagaagttc tcctcgaccg
     2461 gtcgatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     2521 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2581 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2641 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2701 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2761 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2821 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2881 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2941 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     3001 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgaatt ggagcgacct
     3061 catgctatac ctgagaaagc aacctgacct acaggaaaga gttactcaag aataagaatt
     3121 ttcgttttaa aacctaagag tcactttaaa atttgtatac acttattttt tttataactt
     3181 atttaataat aaaaatcata aatcataaga aattcgctta tttagaagtg tcaacaacgt
     3241 atctaccaac gatttgaccc ttttccatct tttcgtaaat ttctggcaag gtagacaagc
     3301 cgacaacctt gattggagac ttgaccaaac ctctggcgaa gaattgttaa ttaagagctc
     3361 agatcttatc gtcgtcatcc ttgtaatcca tcgatactag tgcggccgcc ctttagtgag
     3421 ggttgaattc gaattttcaa aaattcttac tttttttttg gatggacgca aagaagttta
     3481 ataatcatat tacatggcat taccaccata tacatatcca tatacatatc catatctaat
     3541 cttacttata tgttgtggaa atgtaaagag ccccattatc ttagcctaaa aaaaccttct
     3601 ctttggaact ttcagtaata cgcttaactg ctcattgcta tattgaagta cggattagaa
     3661 gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc ctcgtcttca
     3721 ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa caataaagat
     3781 tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc tggccccaca
     3841 aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat tagtttttta
     3901 gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt aacagatata
     3961 taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg gtttgtatta
     4021 cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc tctatacttt
     4081 aacgtcaagg agaaaaaacc ccggatatgc acataactaa tttgggattg catcaggttt
     4141 catttcaaag cggagattcc tataaaggcg cagaagaaac cggaaagcac aaaggtgtaa
     4201 gcgtgatttc atatcaaaga gttaaaaatg gagaaagaaa taaggggatc gaagcgctta
     4261 atcgattata tttacagaac caaacaagct taacaggaaa aagccttttg tttgcgcgtg
     4321 acaaagctga agttttttgc gaggcaataa agttggcagg tggtgatacg tcaaaaatta
     4381 aagccatgat ggaacgatta gatacctata aacttggtga agttaataaa agacatatta
     4441 atgagcttaa taaagtaata agtgaagaga taagagcaca gctaggcatt aaaaataaaa
     4501 aggaattgca aacgaaaata aaacaaatat ttactgatta tttaaataat aagaattggg
     4561 gaccggtcaa caagaatatt agtcatcatg ggaaaaatta tagttttcaa ttaactcctg
     4621 cctctcatat gaagataggg aataaaaata tatttgtcaa agagtataat ggaaaaggaa
     4681 tttgttgtgc ttctacgaga gagcgtgatc atatcgcgaa tatgtggctt tcaaaagtgg
     4741 tggatgacga ggggaaggaa attttttcgg gtatcagaca tggagttata tcagcctatg
     4801 gattgaaaaa aaactcatct gaaagagctg ttgctgctcg taataaagct gaggagttag
     4861 taagcgcagc attatatagc agaccagagt tattatcaca ggctttgtct ggtaaaacag
     4921 tagatttaaa gattgtttcg acttctcttc tgacgccgac cagtttaacc gggggagagg
     4981 agagtatgtt aaaagatcag gtgagcgcat taaaagggtt aaactctaaa aggggggggc
     5041 caacgaaatt attaattcgg aatagtgatg gtcttctgaa agaagtaagc gttaatctaa
     5101 aagtggtgac atttaacttt ggtgtaaatg aattagcgct aaaaatgggc ttaggctgga
     5161 ggaatgttga caagcttaat gatgaatcaa tatgttcttt gttgggggat aatttcctca
     5221 aaaatggtgt gattggaggc tgggctgcag aagcaataga aaaaaatcca ccatgtaaga
     5281 atgatgtgat atatctggct aaccagataa aagagattgt aaacaataag ttacaaaaaa
     5341 atgataatgg agagccgtat aaattgtcac aaagagtgac tctattggct tatactatag
     5401 gtgctgtacc ttgctggaat tgtaagagtg ggaaggacag aacaggtatg caggatgctg
     5461 aaattaaaag agaaataata agaaaacatg aaacaggtca gttttctcaa ttaaatagta
     5521 aattatcctc agaagagaaa agattatttt ctactattct aatgaatagt ggtaatatgg
     5581 aaatccaaga gatgaatact ggtgtgcccg gaaataaagt catgaaaaaa ttgccgctat
     5641 cttcgttaga gctatcttat tctgaaagaa taggggaccc aaaaatatgg aatatggtga
     5701 aagggtattc gtcatttgta taagcttggt accgcggcta gctaagatcc gctctaaccg
     5761 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     5821 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     5881 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcc
     5941 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6001 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6061 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6121 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6601 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6901 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6961 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7021 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7081 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7141 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7201 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7261 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7321 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7381 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7441 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7501 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7561 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7621 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7681 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7741 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7801 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7861 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7921 tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa cgcgagagcg
     7981 ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaaa
     8041 gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa tgcaacgcga
     8101 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     8161 cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac aaaaatgcat
     8221 cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc ctttgtgcgc
     8281 tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt tagaagaagg
     8341 ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc ccgcgtttac
     8401 tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc gattatattc
     8461 tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg atgattcttc
     8521 attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta cgtataggaa
     8581 atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac tacaattttt
     8641 ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag tttagatgca
     8701 agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca gagatatata
     8761 gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt ttagtagctc
     8821 gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc ttttggtttt
     8881 caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc
     8941 aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca catacagctc
     9001 actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca tgagaagaac
     9061 ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat gtaggatgaa
     9121 aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg tatgcttcct
     9181 tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt agtctcatcc
     9241 ttcaatgcta tcatttcctt tgatattgga tcatactaag aaaccatta
//
