LOCUS       sequence-399164-        5855 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 5855)
  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 5855)
  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-Mss4-Kina
FEATURES             Location/Qualifiers
     source          1..5855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      632..641
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             638..1339
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     polyA_signal    2668..2789
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2705..2724)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2759..2778
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2796..3251)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2933..2954)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3145..3164
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3278..3382
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3384..3741
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3384..3575
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3407..3427)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3592..3727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3776..4570
                     /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             3776..4570
                     /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(3830..3849)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4440..4459
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4758..4777)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4802..4849
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5178..5766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5667..5686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      601 tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcgagc tgattaagga
      661 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
      721 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
      781 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
      841 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
      901 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
      961 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     1021 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     1081 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     1141 gagccatctg atcgcaaacg ccaagaccac atatagatcc aagaaacccg ctaagaacct
     1201 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     1261 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     1321 actggggcac aagcttaatt ccggactcag atctcgagct caagcttcga attctgcagt
     1381 cgacggtacc gcgaacaagg tctcagaggg tcacgtgaat ttcatcattg cctacaacat
     1441 gctcactggc ataagagttg ccgtcagtag atgttctggg atcatgaagc cactcacccc
     1501 agcagatttt aggtttacca aaaaactggc ctttgattat cacggcaatg agcttactcc
     1561 gtcatcccag tatgcattta agttcaagga ttattgtccc gaggttttcc gggagcttag
     1621 agcactgttt ggcctcgatc ctgccgatta tctggtgtct ctcacatcta aatacatcct
     1681 ctcagaactg aatagcccag gaaaatctgg gtcatttttt tattatagtc gcgattacaa
     1741 gtatattatt aagactattc accactctga acacatccac ctccggaaac acatccaaga
     1801 atactataac catgtccgcg ataacccgaa tactcttatt tgccaatttt atggcctgca
     1861 tcgcgtcaag atgccaatct ccttccagaa caaaatcaaa catcgaaaaa tatatttcct
     1921 ggtgatgaac aacctgttcc ccccacatct tgatattcac atcacctatg atctcaaggg
     1981 atccacatgg ggcaggttca ccaatcttga caaagagcgc ttggctaaag atcgcagcta
     2041 ccgcccagtg atgaaagacc ttaactggct ggaggagggt caaaagatta agttcgggcc
     2101 actgaagaaa aagacattcc ttacccagct taagaaggat gtagagcttc tggccaaact
     2161 gaacacaatg gattacagcc tgcttattgg catccacgat atcaataaag ccaaagagga
     2221 cgacttgcaa ttggcggaca ctgctagcat tgaagaacag cctcaaacgc agggccctat
     2281 taggacaggc acaggcacag ttgtacggca tttcttccgc gagttcgagg gcggcataag
     2341 agctagcgac cagtttaaca acgacgtcga ccttatttat tatgtgggga taatcgactt
     2401 tctgacaaac tacagtgtca tgaaaaaact cgagactttt tggcgctcac ttcgacacga
     2461 cactaaattg gtcagcgcta tacctccacg ggattatgct aatcgctttt acgagtttat
     2521 cgaggattcc gtctaacggg atccaccgga tctagataac tgatcataat cagccatacc
     2581 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2641 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2701 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2761 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     2821 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2881 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2941 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3001 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3061 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3121 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3181 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3241 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3301 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3361 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3421 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3481 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3601 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3661 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3721 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3781 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3841 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3901 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     3961 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4021 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4081 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4141 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4201 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4261 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4321 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4381 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4441 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4501 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4561 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4621 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4681 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4741 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4801 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     4861 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     4921 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     4981 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5041 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5101 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5161 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5221 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5281 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5341 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5401 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5461 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5521 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5581 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5641 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5701 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5761 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5821 cacatgttct ttcctgcgtt atcccctgat tctgt
//
