LOCUS       sequence-360325-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PtdIns(3,4)P2 biosensor for expression in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: pUG36-NES-EGFP-cPHx3
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2003..2250)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2216..2234
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     2252..2268
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2253..2269
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(3348..4058,4059..4061,4062..4064))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(3348..4058,4059..4061,4062..4064))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3390..3411)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3739..3758
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3998..4019
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4061..4070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4128..4477)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     complement(4528..4548)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4528..4546)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4567..4583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4567..4583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4580..4602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4591..4607
                     /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(4615..4621,4622..4639,4640..4645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4660..4681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4798..4815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4969..5557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5049..5068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5728..6519,6520..6588))
                     /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     6351..6370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6589..6693)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6730..7233
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7274..7292
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     1981 tagggcgaat tgggtaccgg ccgcaaatta aagccttcga gcgtcccaaa accttctcaa
     2041 gcaaggtttt cagtataatg ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa
     2101 tttgaaatat aaataacgtt cttaatacta acataactat aaaaaaataa atagggacct
     2161 agacttcagg ttgtctaact ccttcctttt cggttagagc ggatgtgggg ggagggcgtg
     2221 aatgtaagcg tgacataact aattacatga ctcgaggtcg acggtatcga taagcttgat
     2281 atcgaattcg taccgtcgac tcagcgagag gctgtggaat gtgaggtggc ggtggctgcg
     2341 ttggtaggac ggaaagcgtg tttggattct gaaggaccgg ggggatgctc agaagacgca
     2401 gatctgccgg gaccccgctg tgctacaatg gcgccagaga ctgctttaat ccaactgtgc
     2461 atctcttcag ggctatcagc ctgcacatag aaagttcgag acgttgttac aatttcaaag
     2521 aggttgtccc tcatcattat gtcgctttgc ttacattcct ggactttatg aacctcttta
     2581 agtggtatta cacgaagagg ttccttttcc agttcagatt tgaagtagcc tattgtgttt
     2641 tcatccaatt gaaaatatct tctcttccag tttttcatca ctgctccttg ttttacacaa
     2701 tatccagctt tgataaccgc actatcttga ggtggtttag gagtaaagta aggaagatgg
     2761 ctttgtgacc gtttcagatt atttcttccg ccggaacccc ctgagcctcc gcgagaggct
     2821 gtggaatgtg aggtggcggt ggctgcgttg gtaggacgga aagcgtgttt ggattctgaa
     2881 ggaccggggg gatgctcaga agacgcagat ctgccgggac cccgctgtgc tacaatggcg
     2941 ccagagactg ctttaatcca actgtgcatc tcttcagggc tatcagcctg cacatagaaa
     3001 gttcgagacg ttgttacaat ttcaaagagg ttgtccctca tcattatgtc gctttgctta
     3061 cattcctgga ctttatgaac ctctttaagt ggtattacac gaagaggttc cttttccagt
     3121 tcagatttga agtagcctat tgtgttttca tccaattgaa aatatcttct cttccagttt
     3181 ttcatcactg ctccttgttt tacacaatat ccagctttga taaccgcact atcttgaggt
     3241 ggtttaggag taaagtaagg aagatggctt tgtgaccgtt tcagattatt tctcgagctc
     3301 attttccagg tagaattcga agcttgagct cgagatctga gtccggactt gtacagctcg
     3361 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc
     3421 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc
     3481 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg
     3541 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg
     3601 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg
     3661 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     3721 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     3781 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     3841 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     3901 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     3961 tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     4021 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc gaccggtgcc
     4081 tcatccagct caagctcttc caactttttc tgcagagcca ttctagagta tggatggggg
     4141 taatagaatt gtatctatgt atctgacgac cctgtattac acgaaggaaa tagtagatga
     4201 aaagacaaga gagcaagaaa aaggaaaaac ttcctttcta acagacgctt tacttaacct
     4261 tatatatatc ttattttttt catcgagcgt gttcaattgg acaaggtgcc accttttcga
     4321 cacttcggtt attatgttac acagttttca tgaggatggc gccttgacta acttaattag
     4381 tcatttgcca accaccacag ttccccaata tcgacagctt cacgtgccat ttgccatttt
     4441 gcgatcatgt gacctcaaga gaaaaagcaa aaaataatga gagctaaggg attcgaaccc
     4501 ttgcatccgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4561 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4621 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4741 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4801 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4861 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4921 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4981 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5041 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5101 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5161 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5221 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5281 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5341 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5401 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5461 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5521 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5581 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5641 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5701 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5761 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5821 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5881 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5941 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6001 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6061 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6121 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6181 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6241 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6301 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6361 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6421 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6481 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6541 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6601 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6661 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg
     6721 gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat ataaatatat
     6781 aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg ttttccgaag
     6841 atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc tcttcctgct
     6901 ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca cacgaaaatc
     6961 ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat ctgcttttct
     7021 tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa cctttgttta
     7081 tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta aaatccaaat
     7141 acaaaacata aaaataaata aacacagagt aaattcccaa attattccat cattaaaaga
     7201 tacgaggcgc gtgtaagtta caggcaagcg atccgtctaa gaaaccatta ttatcatgac
     7261 attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
