LOCUS       sequence-360320-        7384 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 7384)
  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 7384)
  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: pUG34-NES-EGFP-cPHx3
FEATURES             Location/Qualifiers
     source          1..7384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(452..639)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(786..805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     905..927
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(965..983)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1031..1534
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        1564..1668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1669..1737,1738..2529)
                     /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(1887..1906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2700..3288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3189..3208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3442..3459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3576..3597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3612..3617,3618..3635,3636..3642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3650..3666
                     /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     3655..3677
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3674..3690
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3674..3690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3709..3729
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3711..3729
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3780..4129
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     regulatory      4187..4196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4193..4195,4196..4198,4199..4909)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4193..4195,4196..4198,4199..4909)
                     /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(4238..4259)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4499..4518)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4846..4867
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5988..6004)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5989..6005)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      6007..6254
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6023..6041)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6272..6291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6273..6291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6301..6318)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6301..6317)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6310..6332)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6458..6913
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6545..6564)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6755..6776
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(7173..7384,1..451))
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
ORIGIN
        1 tctccctttg caaaccaagt tcgacaactg cgtacggcct gttcgaaaga tctaccaccg
       61 ctctggaaag tgcctcatcc aaaggcgcaa atcctgatcc aaaccttttt actccacgca
      121 cggcccctag ggcctcttta aaagcttgac cgagagcaat cccgcagtct tcagtggtgt
      181 gatggtcgtc tatgtgtaag tcaccaatgc actcaacgat tagcgaccag ccggaatgct
      241 tggccagagc atgtatcata tggtccagaa accctatacc tgtgtggacg ttaatcactt
      301 gcgattgtgt ggcctgttct gctactgctt ctgcctcttt ttctgggaag atcgagtgct
      361 ctatcgctag gggaccaccc tttaaagaga tcgcaatctg aatcttggtt tcatttgtaa
      421 tacgctttac tagggctttc tgctctgtca tctttgcctt cgtttatctt gcctgctcat
      481 tttttagtat attcttcgaa gaaatcacat tactttatat aatgtataat tcattatgtg
      541 ataatgccaa tcgctaagaa aaaaaaagag tcatccgcta ggtggaaaaa aaaaaatgaa
      601 aatcattacc gaggcataaa aaaatataga gtgtactaga ggaggccaag agtaatagaa
      661 aaagaaaatt gcgggaaagg actgtgttat gacttccctg actaatgccg tgttcaaacg
      721 atacctggca gtgactccta gcgctcacca agctcttaaa acgggaattt atggtgcact
      781 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
      841 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
      901 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
      961 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1021 ggtccttttc atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata
     1081 taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa
     1141 gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc
     1201 tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat
     1261 cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc
     1321 ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt
     1381 attttttttt cttcattccg taactcttct accttcttta tttactttct aaaatccaaa
     1441 tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag
     1501 atacgaggcg cgtgtaagtt acaggcaagc gatccgtcag gtggcacttt tcggggaaat
     1561 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1621 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     1681 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1741 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     1801 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     1861 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     1921 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     1981 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2041 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2101 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2161 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2221 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2281 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2341 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2401 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2461 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2521 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2581 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2641 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2701 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2761 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2821 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2881 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2941 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3001 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3061 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3361 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3421 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     3481 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3541 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     3601 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3661 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
     3721 cactaaaggg aacaaaagct ggagctccgg atgcaagggt tcgaatccct tagctctcat
     3781 tattttttgc tttttctctt gaggtcacat gatcgcaaaa tggcaaatgg cacgtgaagc
     3841 tgtcgatatt ggggaactgt ggtggttggc aaatgactaa ttaagttagt caaggcgcca
     3901 tcctcatgaa aactgtgtaa cataataacc gaagtgtcga aaaggtggca ccttgtccaa
     3961 ttgaacacgc tcgatgaaaa aaataagata tatataaggt taagtaaagc gtctgttaga
     4021 aaggaagttt ttcctttttc ttgctctctt gtcttttcat ctactatttc cttcgtgtaa
     4081 tacagggtcg tcagatacat agatacaatt ctattacccc catccatact ctagaatggc
     4141 tctgcagaaa aagttggaag agcttgagct ggatgaggca ccggtcgcca ccatggtgag
     4201 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     4261 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     4321 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     4381 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     4441 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     4501 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4561 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4621 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4681 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4741 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4801 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4861 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag
     4921 atctcgagct caagcttcga attctacctg gaaaatgagc tcgagaaata atctgaaacg
     4981 gtcacaaagc catcttcctt actttactcc taaaccacct caagatagtg cggttatcaa
     5041 agctggatat tgtgtaaaac aaggagcagt gatgaaaaac tggaagagaa gatattttca
     5101 attggatgaa aacacaatag gctacttcaa atctgaactg gaaaaggaac ctcttcgtgt
     5161 aataccactt aaagaggttc ataaagtcca ggaatgtaag caaagcgaca taatgatgag
     5221 ggacaacctc tttgaaattg taacaacgtc tcgaactttc tatgtgcagg ctgatagccc
     5281 tgaagagatg cacagttgga ttaaagcagt ctctggcgcc attgtagcac agcggggtcc
     5341 cggcagatct gcgtcttctg agcatccccc cggtccttca gaatccaaac acgctttccg
     5401 tcctaccaac gcagccaccg ccacctcaca ttccacagcc tctcgcggag gctcaggggg
     5461 ttccggcgga agaaataatc tgaaacggtc acaaagccat cttccttact ttactcctaa
     5521 accacctcaa gatagtgcgg ttatcaaagc tggatattgt gtaaaacaag gagcagtgat
     5581 gaaaaactgg aagagaagat attttcaatt ggatgaaaac acaataggct acttcaaatc
     5641 tgaactggaa aaggaacctc ttcgtgtaat accacttaaa gaggttcata aagtccagga
     5701 atgtaagcaa agcgacataa tgatgaggga caacctcttt gaaattgtaa caacgtctcg
     5761 aactttctat gtgcaggctg atagccctga agagatgcac agttggatta aagcagtctc
     5821 tggcgccatt gtagcacagc ggggtcccgg cagatctgcg tcttctgagc atccccccgg
     5881 tccttcagaa tccaaacacg ctttccgtcc taccaacgca gccaccgcca cctcacattc
     5941 cacagcctct cgctgagtcg acggtacgaa ttcgatatca agcttatcga taccgtcgac
     6001 ctcgagtcat gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc
     6061 taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag
     6121 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac
     6181 gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga
     6241 aggctttaat ttgcggccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     6301 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     6361 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     6421 cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta
     6481 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     6541 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6601 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6661 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6721 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6781 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     6841 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6901 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6961 gcatagatcc gtcgagttca agagaaaaaa aaagaaaaag caaaaagaaa aaaggaaagc
     7021 gcgcctcgtt cagaatgaca cgtatagaat gatgcattac cttgtcatct tcagtatcat
     7081 actgttcgta tacatactta ctgacattca taggtataca tatatacaca tgtatatata
     7141 tcgtatgctg cagctttaaa taatcggtgt cactacataa gaacaccttt ggtggaggga
     7201 acatcgttgg taccattggg cgaggtggct tctcttatgg caaccgcaag agccttgaac
     7261 gcactctcac tacggtgatg atcattcttg cctcgcagac aatcaacgtg gagggtaatt
     7321 ctgctagcct ctgcaaagct ttcaagaaaa tgcgggatca tctcgcaaga gagatctcct
     7381 actt
//
