LOCUS       sequence-399478-        6354 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 6354)
  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 6354)
  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: EGFP-PIP5K1BD266K
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             80..874
                     /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             80..874
                     /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(134..153)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     744..763
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1062..1081)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1106..1153
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1482..2070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1971..1990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2245..2548
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2549..2752
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2703..2723
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2791..2800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2797..2799,2800..2802,2803..3513)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2797..2799,2800..2802,2803..3513)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2842..2863)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3103..3122)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3450..3471
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5326..5447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5363..5382)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5417..5436
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5454..5909)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5591..5612)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5803..5822
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5936..6040
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6042..6354,1..45)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6042..6233
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6065..6085)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6250..6354,1..31)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
       61 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
      121 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
      181 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
      241 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
      301 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
      361 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
      421 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
      481 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
      541 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
      601 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
      661 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
      721 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
      781 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
      841 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
      901 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
      961 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     1021 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     1081 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     1141 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     1201 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     1261 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     1321 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1381 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1441 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1501 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1561 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1621 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1681 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1741 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1801 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1861 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1921 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1981 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2041 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2101 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2161 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     2221 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2281 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2341 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2401 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2461 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2521 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     2581 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     2641 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     2701 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     2761 gaaccgtcag atccgctagc gctaccggtc gccaccatgg tgagcaaggg cgaggagctg
     2821 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2881 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2941 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3001 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3061 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3121 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3181 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3241 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3301 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3361 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     3421 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3481 gggatcactc tcggcatgga cgagctgtac aagtccggac tcagatctcg agctcaagct
     3541 tcgaattctg cagtcgacgg taccgcgatg tcttctgctg ctgaaaatgg agaggcagca
     3601 cctggaaaac aaaatgaaga aaaaacctat aaaaagactg catcatctgc tattaaaggt
     3661 gctattcagc tgggaatagg atacacagtg ggtaatctca cttccaagcc agaacgagat
     3721 gttcttatgc aagactttta tgtggtggaa agtgtgttcc tacccagcga agggagcaat
     3781 ctgaccccag cacatcacta cccagacttt agatttaaga catacgctcc attagcattc
     3841 cgatatttca gagaactttt tggtatcaag cctgatgatt acttgtattc catctgcagt
     3901 gaacctctaa tagaactgtc taaccctgga gccagtggat ccttgttttt tgtgaccagt
     3961 gatgatgaat ttatcatcaa aacagttcag cacaaagaag ctgagtttct tcagaagcta
     4021 ctgccaggct attacatgaa tttaaaccag aatccaagga ctcttttgcc aaaattttac
     4081 ggactgtatt gtatgcaatc aggaggcatt aatatcagga ttgtggtgat gaacaacgtt
     4141 ttgccacgct ccatgagaat gcactttaca tatgacttga aaggctcaac gtataagcga
     4201 agagcatccc gtaaagagag agagaaatcc aaccccacat ttaaggactt agatttcctg
     4261 caagacatgc acgaagggtt gtattttgat acggaaacat acaacgcgct tatgaaaaca
     4321 cttcagagag actgccgggt gctagaaagc ttcaagatca tgaagtatag ccttctgttg
     4381 ggaattcatt tcctggacca ttccctcaaa gagaaagagg aggagacccc acaaaatgtg
     4441 cctgatgcta agcggactgg gatgcagaag gttctctact caacagccat ggaatctatc
     4501 cagggtccag ggaaatctgg agatgggata atcacagaga acccagacac aatgggaggc
     4561 attccagcta aaagccatag gggagaaaaa ctacttttat ttatgggcat tattgacatt
     4621 ctgcaatcat ataggttaat gaagaagtta gaacattcct ggaaagctct tgtttatgat
     4681 ggggacactg tttctgttca tagaccaagc ttttatgcag acagatttct taagttcatg
     4741 aattccagag ttttcaagaa aattcaagct ttgaaggctt caccgtctaa gaaacggtgc
     4801 aattcaatcg ccgccctaaa ggccacttca caggagattg tgtcctcaat tagccaggaa
     4861 tggaaggatg agaagcggga tttgctgact gaaggacaaa gttttagcag ccttgatgaa
     4921 gaagccctgg gatcccgaca caggccagac ctggtcccta gcactccatc actgtttgaa
     4981 gctgcttcct tggcaaccac aatttcatct tcttccttat acgtcaatga gcactatcca
     5041 cacgacaggc ctacactcta ttcaaacagc aaagggttac cttccagttc aacatttacc
     5101 ttggaagagg ggaccatcta cttgaccgct gagcccaaca ctctggaagt gcaggatgac
     5161 aatgcttctg tgcttgacgt ctatttataa cgcccgggat ccaccggatc tagataactg
     5221 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     5281 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     5341 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5401 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     5461 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5521 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5581 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5641 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5701 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5761 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5821 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5881 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     5941 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6001 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     6061 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     6121 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     6181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     6241 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     6301 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attc
//
