LOCUS       sequence-356063-        6374 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 6374)
  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 6374)
  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..6374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(173..988)
                     /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(173..988)
                     /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     896..915
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     980..999
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1110..1698
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1599..1618
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1852..1869
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1884..2026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1952..1971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2112..2134
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2128..2319)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3128..4210)
                     /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     4172..4191
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4211..4288)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4494..4513
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4597..4616
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4597..4615
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4616..4640
                     /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             4655..4677
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4696..4713
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4723..4740
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(4747..4749,4750..4752,4753..5463)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4747..4749,4750..4752,4753..5463)
                     /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(4792..4813)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5053..5072)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5400..5421
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5899..5917)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5913..5960
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5997..6374,1..80)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6084..6103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6294..6315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
       61 aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      121 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
      181 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
      241 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
      301 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
      361 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
      421 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
      481 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
      541 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
      601 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
      661 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
      721 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
      781 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
      841 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
      901 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
      961 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     1021 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1081 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1141 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1201 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1261 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1321 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1381 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1441 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1501 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1561 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1621 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1681 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1741 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     1801 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     1861 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1921 atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     1981 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     2041 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     2101 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     2161 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     2221 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     2281 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     2341 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     2401 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     2461 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     2521 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     2581 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     2641 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     2701 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     2761 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     2821 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     2881 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     2941 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     3001 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     3061 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     3121 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3181 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     3241 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     3301 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     3361 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     3421 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     3481 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     3541 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3601 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3661 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     3721 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     3781 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     3841 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     3901 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     3961 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     4021 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     4081 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     4141 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     4201 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     4261 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     4321 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     4381 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     4441 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     4501 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     4561 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     4621 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     4681 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
     4741 ggaggtatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4801 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     4861 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     4921 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     4981 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     5041 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     5101 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     5161 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     5221 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     5281 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     5341 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     5401 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     5461 aagtccggac tcagatctcg agctcaagct ggttccggcc acggcctaca ggatgatgag
     5521 gatctacagg cgctgctgaa gggcagccag ctcctgaagg tgaagtccag ctcatggagg
     5581 agagagcgct tctacaagtt gcaggaggac tgcaagacca tctggcagga gtcccgcaag
     5641 gtcatgcgga ccccggagtc ccagctgttc tccatcgagg acattcagga ggtgcgaatg
     5701 gggcaccgca cggagggtct ggagaagttc gcccgtgatg tgcccgagga ccgctgcttc
     5761 tccattgtct tcaaggacca gcgcaataca ctagacctca tcgccccatc gccagctgat
     5821 gcccagcact gggtgctggg gctgcacaag atcatccacc actcaggctc catggaccag
     5881 cgtcagaagt gagtaccacc gctgagcaat aactagcata accccttggg gcctctaaac
     5941 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     6001 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6061 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6121 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6181 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6241 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6301 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6361 gattttgccg atta
//
