LOCUS       sequence-360273-        6368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PtdIns(4,5)P2 biosensor for purification from bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6368)
  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 6368)
  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: His6-EGFP-PH-PLCD1
FEATURES             Location/Qualifiers
     source          1..6368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(164..979)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(164..979)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     887..906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     971..990
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1101..1689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1590..1609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1843..1860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1875..2014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1940..1959)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2100..2122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2116..2307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3116..4198)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4160..4179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4199..4276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4482..4501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4585..4604
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4585..4603
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4604..4628
                     /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)."
     RBS             4643..4665
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4684..4701
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4711..4728
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(4735..4737,4738..4740,4741..5451)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4735..4737,4738..4740,4741..5451)
                     /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(4780..4801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5041..5060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5388..5409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5887..5905)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5901..5948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5985..6368,1..72)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6072..6091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6282..6303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
       61 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      121 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
      181 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
      241 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
      301 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
      361 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
      421 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
      481 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
      541 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
      601 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
      661 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
      721 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
      781 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
      841 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
      901 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
      961 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     1021 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     1261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     1441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     1501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     1561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     1621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     1681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     1741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     1801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     1861 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     1921 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     1981 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2041 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2101 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     2161 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     2221 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     2281 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     2341 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     2401 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     2461 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     2521 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     2581 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     2641 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     2701 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     2761 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     2821 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     2881 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     2941 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     3001 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     3061 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3121 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3181 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     3241 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     3301 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     3361 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     3421 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     3481 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     3541 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     3601 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     3661 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     3721 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     3781 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     3841 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     3901 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     3961 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     4021 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     4081 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4141 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4201 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     4261 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     4321 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     4381 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     4441 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     4501 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     4561 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     4621 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggcagc
     4681 agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcgg aggtatggtg
     4741 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     4801 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     4861 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     4921 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     4981 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     5041 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     5101 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     5161 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     5221 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     5281 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     5341 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     5401 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     5461 agatctcgag ctcaagctgg ttccggccac ggcctacagg atgatgagga tctacaggcg
     5521 ctgctgaagg gcagccagct cctgaaggtg aagtccagct catggaggag agagcgcttc
     5581 tacaagttgc aggaggactg caagaccatc tggcaggagt cccgcaaggt catgcggacc
     5641 ccggagtccc agctgttctc catcgaggac attcaggagg tgcgaatggg gcaccgcacg
     5701 gagggtctgg agaagttcgc ccgtgatgtg cccgaggacc gctgcttctc cattgtcttc
     5761 aaggaccagc gcaatacact agacctcatc gccccatcgc cagctgatgc ccagcactgg
     5821 gtgctggggc tgcacaagat catccaccac tcaggctcca tggaccagcg tcagaagtga
     5881 gtaccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5941 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     6001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6061 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6181 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6301 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6361 ttcggcct
//
