LOCUS       sequence-399287-        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-PIP4K2AD273K
FEATURES             Location/Qualifiers
     source          1..5922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        135..438
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        439..642
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     593..613
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      681..690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             687..1388
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     polyA_signal    2784..2905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2821..2840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2875..2894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2912..3367)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3261..3280
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3394..3498
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3500..3857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3500..3691
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3523..3543)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3708..3843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3892..4686
                     /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             3892..4686
                     /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(3946..3965)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4556..4575
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4874..4893)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4918..4965
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5294..5882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5783..5802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
       61 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      121 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      181 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      241 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      301 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      361 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      421 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      481 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      541 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      601 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      661 atccgctagc gctaccggtc gccaccatgg tgagcgagct gattaaggag aacatgcaca
      721 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
      781 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
      841 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
      901 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
      961 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     1021 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     1081 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     1141 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     1201 tcgcaaacgc caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     1261 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     1321 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1381 agcttaattc cggactcaga tctcgagctc aagcttcgaa ttctgcagtc gacatggcga
     1441 cccccggcaa cctagggtcc tccgtcctgg cgagcaagac caagaccaag aagaagcact
     1501 tcgtagcgca gaaagtgaag ctgtttcggg ccagcgaccc gctgctcagc gtcctcatgt
     1561 ggggggtaaa ccactcgatc aatgaactga gccatgttca aatccctgtt atgttgatgc
     1621 cagatgactt caaagcctat tcaaaaataa aggtggacaa tcaccttttt aacaaagaaa
     1681 acatgccgag ccatttcaag tttaaggaat actgcccgat ggtcttccgt aacctgcggg
     1741 agaggtttgg aattgatgat caagatttcc agaattccct gaccaggagc gcacccctcc
     1801 ccaacgactc ccaggcccgc agtggagctc gttttcacac ttcctacgac aaaagataca
     1861 tcatcaagac tattaccagt gaagacgtgg ccgaaatgca caacatcctg aagaaatacc
     1921 accagtacat agtggaatgt catgggatca cccttcttcc ccagttcttg ggcatgtacc
     1981 ggcttaatgt tgatggagtt gaaatatatg tgatagttac aagaaatgta ttcagccacc
     2041 gtttgtctgt gtataggaaa tacgacttaa agggctctac agtggctaga gaagctagtg
     2101 acaaagaaaa ggccaaagaa ctgccaactc tgaaagataa tgatttcatt aatgagggcc
     2161 aaaagattta tattgatgac aacagcaaga aggtcttcct ggaaaaacta aaaaaggatg
     2221 ttgagtttct ggcccagctg aagctcatga aatacagtct gctggtggga attcatgatg
     2281 tggagagagc cgaacaggag gaagtggagt gtgaggagaa cgatggggag gaggagggcg
     2341 agagcgatgg cacccacccg gtgggaaccc ccccagatag ccccgggaat acactgaaca
     2401 gctcaccacc cctggctccc ggggagttcg atccgaacat cgacgtctat ggaattaagt
     2461 gccatgaaaa ctcgcctagg aaggaggtgt acttcatggc aattattgac atccttactc
     2521 attatgatgc aaaaaagaaa gctgcccatg ctgcaaaaac tgttaaacat ggcgctggcg
     2581 cggagatctc caccgtgaac ccagaacagt attcaaagcg ctttttggac tttattggcc
     2641 acatcttgac gtaaggatcc accggatcta gataactgat cataatcagc cataccacat
     2701 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     2761 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2821 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2881 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     2941 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3001 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3061 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3121 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3181 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3241 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3301 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3361 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3421 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3481 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3541 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3601 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3661 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3721 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3781 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3841 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3901 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3961 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4021 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     4081 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4141 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4201 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4261 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4321 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4381 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     4441 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4501 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4561 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4621 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4681 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4741 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4801 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4861 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4921 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4981 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     5041 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     5101 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     5161 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5221 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5281 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5341 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5401 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5461 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5521 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5581 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5641 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5701 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5761 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5821 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5881 aaaacgccag caacgcggcc tttttacggt tcctggcctt tt
//
