LOCUS       sequence-399276-        5952 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 5952)
  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 5952)
  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- PIP4K2BD278K
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        98..401
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        402..605
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     556..576
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      644..653
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             650..1351
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     polyA_signal    2777..2898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2814..2833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2868..2887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2905..3360)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3042..3063)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3254..3273
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3387..3491
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3493..3850
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3493..3684
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3516..3536)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3701..3836
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3885..4679
                     /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             3885..4679
                     /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(3939..3958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4549..4568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4867..4886)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4911..4958
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5287..5875
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5776..5795
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
       61 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      121 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      181 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      241 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      301 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      361 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      421 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      481 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      541 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      601 gagctggttt agtgaaccgt cagatccgct agcgctaccg gtcgccacca tggtgagcga
      661 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca
      721 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
      781 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct
      841 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc
      901 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
      961 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     1021 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt
     1081 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa
     1141 gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc
     1201 cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa
     1261 ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga
     1321 cctccctagc aaactggggc acaagcttaa ttccggactc agatctcgag ctcaagcttc
     1381 gaattctgca gtcgacatgt cgtccaactg caccagcacc acggcggtgg cggtggcgcc
     1441 gctcagcgcc agcaagacca agaccaagaa gaagcatttc gtgtgccaga aagtgaagct
     1501 attccgggcc agcgagccga tcctcagcgt cctgatgtgg ggggtgaacc acacgatcaa
     1561 tgagctgagc aatgttcctg ttcctgtcat gctaatgcca gatgacttca aagcctacag
     1621 caagatcaag gtggacaatc atctcttcaa taaggagaac ctgcccagcc gctttaagtt
     1681 taaggagtat tgccccatgg tgttccgaaa ccttcgggag aggtttggaa ttgatgatca
     1741 ggattaccag aattcagtga cgcgcagcgc ccccatcaac agtgacagcc agggtcggtg
     1801 tggcacgcgt ttcctcacca cctacgaccg gcgctttgtc atcaagactg tgtccagcga
     1861 ggacgtggcg gagatgcaca acatcttaaa gaaataccac cagtttatag tggagtgtca
     1921 tggcaacacg cttttgccac agttcctggg catgtaccgc ctgaccgtgg atggtgtgga
     1981 aacctacatg gtggttacca ggaacgtgtt cagccatcgg ctcactgtgc atcgcaagta
     2041 tgacctcaag ggttctacgg ttgccagaga agcgagcgac aaggagaagg ccaaggactt
     2101 gccaacattc aaagacaatg acttcctcaa tgaagggcag aagctgcatg tgggagagga
     2161 gagtaaaaag aacttcctgg agaaactgaa gcgggacgtt gagttcttgg cacagctgaa
     2221 gatcatgaaa tacagcctgc tggtgggcat ccacgacgtg gaccgggcag agcaggagga
     2281 gatggaggtg gaggagcggg cagaggacga ggagtgtgag aatgatgggg tgggtggcaa
     2341 cctactctgc tcctatggca cacctccgga cagccctggc aacctcctca gctttcctcg
     2401 gttctttggt cctggggaat tcgacccctc tgttgacgtc tatgccatga aaagccatga
     2461 aagttccccc aagaaggagg tgtatttcat ggccatcatt gatatcctca cgccatacga
     2521 tacaaagaag aaagctgcac atgctgccaa aacggtgaaa cacggggcag gggccgagat
     2581 ctcgactgtg aaccctgagc agtactccaa acgcttcaac gagtttatgt ccaacatcct
     2641 gacgtaggga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     2701 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2761 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2881 actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     2941 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3001 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3061 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3121 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3181 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3241 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3301 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3361 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3421 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3481 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3541 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3601 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3661 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3721 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3781 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3841 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3901 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3961 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4021 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4081 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4141 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4201 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4261 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4321 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4381 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4441 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4501 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4561 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4621 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4681 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4741 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4801 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     4861 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     4921 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     4981 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5041 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5101 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5161 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5221 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5281 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5341 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5401 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5461 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5521 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5581 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5641 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5701 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5761 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5821 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5881 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5941 tcctgcgtta tc
//
