LOCUS       sequence-399144-        5922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5922)
  AUTHORS   Wills RC, Doyle CP, Zewe JP, Pacheco J, Hansen SD, Hammond GRV
  TITLE     A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at
            the plasma membrane.
  JOURNAL   J Cell Sci. 2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494.
  PUBMED    37534432
REFERENCE   2  (bases 1 to 5922)
  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: "J Cell Sci.
            2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494."
COMMENT     Sequence Label: TagBFP2-PIP4K2A
FEATURES             Location/Qualifiers
     source          1..5922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             154..948
                     /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             154..948
                     /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(208..227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     818..837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1136..1155)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1180..1227
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1556..2144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2045..2064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2319..2622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2623..2826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2777..2797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2865..2874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2871..3572
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     polyA_signal    4968..5089
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5005..5024)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5059..5078
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5096..5551)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5233..5254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5445..5464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5578..5682
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5684..5922,1..119)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5684..5875
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5707..5727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5892..5922,1..105)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
       61 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
      121 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
      181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
      241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
      301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
      361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
      421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
      481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
      541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
      601 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
      661 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
      721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
      781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
      841 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
      901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
      961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     1021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     1081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     1141 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     1201 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     1261 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     1321 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     1381 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     1441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     1501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     1741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2221 cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt
     2281 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     2341 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     2401 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2461 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2521 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2581 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2641 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2701 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2761 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2821 agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc atggtgagcg
     2881 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
     2941 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     3001 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
     3061 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
     3121 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     3181 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     3241 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     3301 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
     3361 agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac
     3421 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     3481 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     3541 acctccctag caaactgggg cacaagctta attccggact cagatctcga gctcaagctt
     3601 cgaattctgc agtcgacatg gcgacccccg gcaacctagg gtcctccgtc ctggcgagca
     3661 agaccaagac caagaagaag cacttcgtag cgcagaaagt gaagctgttt cgggccagcg
     3721 acccgctgct cagcgtcctc atgtgggggg taaaccactc gatcaatgaa ctgagccatg
     3781 ttcaaatccc tgttatgttg atgccagatg acttcaaagc ctattcaaaa ataaaggtgg
     3841 acaatcacct ttttaacaaa gaaaacatgc cgagccattt caagtttaag gaatactgcc
     3901 cgatggtctt ccgtaacctg cgggagaggt ttggaattga tgatcaagat ttccagaatt
     3961 ccctgaccag gagcgcaccc ctccccaacg actcccaggc ccgcagtgga gctcgttttc
     4021 acacttccta cgacaaaaga tacatcatca agactattac cagtgaagac gtggccgaaa
     4081 tgcacaacat cctgaagaaa taccaccagt acatagtgga atgtcatggg atcacccttc
     4141 ttccccagtt cttgggcatg taccggctta atgttgatgg agttgaaata tatgtgatag
     4201 ttacaagaaa tgtattcagc caccgtttgt ctgtgtatag gaaatacgac ttaaagggct
     4261 ctacagtggc tagagaagct agtgacaaag aaaaggccaa agaactgcca actctgaaag
     4321 ataatgattt cattaatgag ggccaaaaga tttatattga tgacaacagc aagaaggtct
     4381 tcctggaaaa actaaaaaag gatgttgagt ttctggccca gctgaagctc atggactaca
     4441 gtctgctggt gggaattcat gatgtggaga gagccgaaca ggaggaagtg gagtgtgagg
     4501 agaacgatgg ggaggaggag ggcgagagcg atggcaccca cccggtggga acccccccag
     4561 atagccccgg gaatacactg aacagctcac cacccctggc tcccggggag ttcgatccga
     4621 acatcgacgt ctatggaatt aagtgccatg aaaactcgcc taggaaggag gtgtacttca
     4681 tggcaattat tgacatcctt actcattatg atgcaaaaaa gaaagctgcc catgctgcaa
     4741 aaactgttaa acatggcgct ggcgcggaga tctccaccgt gaacccagaa cagtattcaa
     4801 agcgcttttt ggactttatt ggccacatct tgacgtaagg atccaccgga tctagataac
     4861 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4921 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     4981 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5041 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     5101 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5161 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5221 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5281 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5341 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5401 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5461 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5521 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     5581 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5641 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     5701 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     5761 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     5821 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     5881 taactccgcc catcccgccc ctaactccgc ccagttccgc cc
//
