LOCUS       sequence-399217-        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-Mss4D636K-Kina
FEATURES             Location/Qualifiers
     source          1..5855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        54..411
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        54..245
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(77..97)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      262..397
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             446..1240
                     /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             446..1240
                     /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(500..519)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1110..1129
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1428..1447)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1472..1519
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1848..2436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2337..2356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2611..2914
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2915..3118
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3069..3089
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3157..3166
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3163..3864
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     polyA_signal    5193..5314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5230..5249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5284..5303
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5321..5776)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5458..5479)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5670..5689
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5803..5855,1..52)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
       61 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
      121 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
      181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      241 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      301 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      361 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
      421 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
      481 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
      541 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
      601 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
      661 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
      721 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
      781 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
      841 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
      901 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
      961 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     1021 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     1081 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     1141 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     1201 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     1261 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     1321 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     1381 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     1441 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     1501 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     1561 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     1621 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     1681 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     1741 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     1801 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1861 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1921 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1981 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2041 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2101 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2161 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2221 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2281 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2341 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2401 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2461 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2521 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
     2581 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     2641 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     2701 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     2761 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     2821 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     2881 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     2941 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     3001 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     3061 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
     3121 tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca ccatggtgag cgagctgatt
     3181 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
     3241 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
     3301 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
     3361 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
     3421 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     3481 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     3541 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag
     3601 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
     3661 ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag
     3721 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     3781 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
     3841 agcaaactgg ggcacaagct taattccgga ctcagatctc gagctcaagc ttcgaattct
     3901 gcagtcgacg gtaccgcgaa caaggtctca gagggtcacg tgaatttcat cattgcctac
     3961 aacatgctca ctggcataag agttgccgtc agtagatgtt ctgggatcat gaagccactc
     4021 accccagcag attttaggtt taccaaaaaa ctggcctttg attatcacgg caatgagctt
     4081 actccgtcat cccagtatgc atttaagttc aaggattatt gtcccgaggt tttccgggag
     4141 cttagagcac tgtttggcct cgatcctgcc gattatctgg tgtctctcac atctaaatac
     4201 atcctctcag aactgaatag cccaggaaaa tctgggtcat ttttttatta tagtcgcgat
     4261 tacaagtata ttattaagac tattcaccac tctgaacaca tccacctccg gaaacacatc
     4321 caagaatact ataaccatgt ccgcgataac ccgaatactc ttatttgcca attttatggc
     4381 ctgcatcgcg tcaagatgcc aatctccttc cagaacaaaa tcaaacatcg aaaaatatat
     4441 ttcctggtga tgaacaacct gttcccccca catcttgata ttcacatcac ctatgatctc
     4501 aagggatcca catggggcag gttcaccaat cttgacaaag agcgcttggc taaagatcgc
     4561 agctaccgcc cagtgatgaa agaccttaac tggctggagg agggtcaaaa gattaagttc
     4621 gggccactga agaaaaagac attccttacc cagcttaaga aggatgtaga gcttctggcc
     4681 aaactgaaca caatgaaata cagcctgctt attggcatcc acgatatcaa taaagccaaa
     4741 gaggacgact tgcaattggc ggacactgct agcattgaag aacagcctca aacgcagggc
     4801 cctattagga caggcacagg cacagttgta cggcatttct tccgcgagtt cgagggcggc
     4861 ataagagcta gcgaccagtt taacaacgac gtcgacctta tttattatgt ggggataatc
     4921 gactttctga caaactacag tgtcatgaaa aaactcgaga ctttttggcg ctcacttcga
     4981 cacgacacta aattggtcag cgctatacct ccacgggatt atgctaatcg cttttacgag
     5041 tttatcgagg attccgtcta acgggatcca ccggatctag ataactgatc ataatcagcc
     5101 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     5161 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     5221 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5281 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     5341 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5401 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5461 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5521 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5581 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5641 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5701 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5761 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5821 ttatttttct aaatacattc aaatatgtat ccgct
//
