LOCUS       sequence-360285-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PtdIns(3,4)P2 biosensor for purification from bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  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 6468)
  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-cPHx1
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..466
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(98..117)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     308..329
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(558..1373)
                     /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(558..1373)
                     /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     1281..1300
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1365..1384
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1495..2083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1984..2003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2237..2254
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2269..2408
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2334..2353)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2494..2516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2510..2701)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3510..4592)
                     /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     4554..4573
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4593..4670)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4876..4895
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4979..4998
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4979..4997
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4998..5022
                     /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             5037..5059
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5078..5095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5105..5122
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5129..5131,5132..5134,5135..5845)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5129..5131,5132..5134,5135..5845)
                     /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(5174..5195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5435..5454)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5782..5803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6381..6399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6395..6442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
       61 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      121 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      181 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
      241 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      301 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
      361 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
      421 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
      481 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
      541 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
      601 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
      661 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
      721 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
      781 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
      841 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
      901 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
      961 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     1021 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     1081 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     1141 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     1201 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     1261 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     1321 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     1381 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     1441 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1501 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1561 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1621 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1681 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1741 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1801 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1861 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1921 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1981 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2041 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2101 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2161 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2221 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     2281 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     2341 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     2401 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2461 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2521 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     2581 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     2641 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     2701 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     2761 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     2821 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     2881 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     2941 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     3001 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     3061 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     3121 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     3181 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     3241 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     3301 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     3361 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     3421 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     3481 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3541 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3601 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     3661 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     3721 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     3781 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     3841 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     3901 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     3961 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     4021 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     4081 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     4141 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     4201 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     4261 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     4321 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     4381 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     4441 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     4501 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     4561 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     4621 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     4681 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     4741 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     4801 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     4861 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     4921 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     4981 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     5041 tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag
     5101 cggcctggtg ccgcgcggca gcggaggtat ggtgagcaag ggcgaggagc tgttcaccgg
     5161 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     5221 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     5281 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     5341 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     5401 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     5461 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     5521 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     5581 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     5641 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     5701 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     5761 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     5821 tctcggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc
     5881 tagaaataat ctgaaacggt cacaaagcca tcttccttac tttactccta aaccacctca
     5941 agatagtgcg gttatcaaag ctggatattg tgtaaaacaa ggagcagtga tgaaaaactg
     6001 gaagagaaga tattttcaat tggatgaaaa cacaataggc tacttcaaat ctgaactgga
     6061 aaaggaacct cttcgtgtaa taccacttaa agaggttcat aaagtccagg aatgtaagca
     6121 aagcgacata atgatgaggg acaacctctt tgaaattgta acaacgtctc gaactttcta
     6181 tgtgcaggct gatagccctg aagagatgca cagttggatt aaagcagtct ctggcgccat
     6241 tgtagcacag cggggtcccg gcagatctgc gtcttctgag catccccccg gtccttcaga
     6301 atccaaacac gctttccgtc ctaccaacgc agccaccgcc acctcacatt ccacagcctc
     6361 tcgctgagtc gacggtacca ccgctgagca ataactagca taaccccttg gggcctctaa
     6421 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatt
//
