LOCUS       sequence-207650-        5097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PI4P biosensor .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5097)
  AUTHORS   Zewe JP, Wills RC, Sangappa S, Goulden BD, Hammond GR
  TITLE     SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic 
            reticulum to maintain a steep chemical gradient with donor 
            membranes.
  JOURNAL   Elife. 2018 Feb 20;7. pii: 35588. doi: 10.7554/eLife.35588.
  PUBMED    29461204
REFERENCE   2  (bases 1 to 5097)
  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: "Elife."; 
            date: "2018-02-20"; volume: "7. pii: 35588. doi"; pages: " 
            10.7554/eLife.35588"
COMMENT     Sequence Label: NES-EGFP-P4Mx1
FEATURES             Location/Qualifiers
     source          1..5097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      594..603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      651..660
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(657..659,660..662,663..1373)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(657..659,660..662,663..1373)
                     /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(702..723)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(963..982)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1310..1331
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1446..1751
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     polyA_signal    1884..2005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1921..1940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1975..1994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2012..2467)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2149..2170)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2361..2380
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2494..2598
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2600..2957
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2600..2791
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2623..2643)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2808..2943
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2992..3786
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2992..3786
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3046..3065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3656..3675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3974..3993)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4018..4065
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4394..4982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4883..4902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagccacca
      601 tggctctgca gaaaaagttg gaagagcttg agctggatga ggcaccggtc gccaccatgg
      661 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      721 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      781 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      841 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      901 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      961 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1021 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1081 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1141 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1201 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1261 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1321 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     1381 tcagatctcg agctcaagct tcgaattctg cagtcgacgg tggtagtgct tccggactca
     1441 gatccacggc aagcacggaa aactttaaaa atgttaaaga aaaatatcag caaatgcgag
     1501 gtgatgcttt aaaaacagaa atcctggctg atttcaagga taaactggct gaagctaccg
     1561 atgaacagag ccttaagcaa attgttgctg aattgaaaag taaagatgaa tatagaattt
     1621 tggctaaggg gcaagggtta acaacccagc ttttagggtt aaagacaagt tcagtgtctt
     1681 catttgagaa aatggttgag gaaacaagag aaagtattaa gtctcaagaa agacaaacca
     1741 ttaagataaa ataaggatcc accggatcta gataactgat cataatcagc cataccacat
     1801 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1861 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1981 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     2041 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2101 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2161 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2221 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2281 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2341 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2401 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2461 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2521 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2581 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2641 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2701 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2761 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2821 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2881 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2941 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3001 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3061 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3121 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3181 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3241 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3301 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3361 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3421 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3481 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3541 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3601 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3661 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3721 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3781 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3841 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3901 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     3961 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4021 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4081 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4141 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4201 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4261 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4321 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4381 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4441 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4501 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4561 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4621 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4681 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4741 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4801 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4861 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4921 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4981 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5041 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcatt
//
