LOCUS       sequence-399173-        6416 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 6416)
  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 6416)
  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-PIP5K1A
FEATURES             Location/Qualifiers
     source          1..6416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             75..869
                     /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             75..869
                     /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(129..148)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     739..758
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1057..1076)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1101..1148
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1477..2065
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1966..1985
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2240..2543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2544..2747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2698..2718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2786..2795
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2792..2794,2795..2797,2798..3508)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2792..2794,2795..2797,2798..3508)
                     /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(2837..2858)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3098..3117)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3445..3466
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5383..5504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5420..5439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5474..5493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5511..5966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5648..5669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5860..5879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5993..6097
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6099..6416,1..40)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6099..6290
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6122..6142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6307..6416,1..26)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
       61 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
      121 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
      181 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
      241 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
      301 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
      361 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
      421 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
      481 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
      541 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
      601 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
      661 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
      721 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
      781 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
      841 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
      901 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
      961 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     1021 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     1081 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     1141 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     1201 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     1261 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     1321 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1381 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1441 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1501 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1561 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1621 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1681 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1741 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1801 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1861 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1921 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1981 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2041 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2101 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2161 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
     2221 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2281 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2341 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2401 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2461 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2521 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2581 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2641 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2701 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
     2761 gtcagatccg ctagcgctac cggtcgccac catggtgagc aagggcgagg agctgttcac
     2821 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2881 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2941 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     3001 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3061 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3121 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3181 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     3241 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     3301 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     3361 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     3421 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     3481 cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagctc aagcttcgaa
     3541 ttctgcagtc gacggtaccg cgatggcgtc ggcctcctcc gggccgtcgt cttcggtcgg
     3601 tttttcatcc tttgatcccg cggtcccttc ctgtaccttg tcctcagcag catctggaat
     3661 caagagaccc atggcatctg aggtcttgga agctagacag gattcttaca tctcattggt
     3721 gccttatgcc tctggcatgc ccatcaagaa aataggccat agaagtgttg attcctcagg
     3781 agagacaaca tataaaaaga caacctcatc agccttgaaa ggtgccatcc agttaggcat
     3841 tacccacact gtggggagcc tgagtaccaa accagagcgt gatgtcctca tgcaagattt
     3901 ctacgtggtt gagagtatct tctttcccag tgaagggagc aacctgaccc ctgctcatca
     3961 ctacaatgac tttcgtttca agacctatgc acctgttgcc ttccgctact tccgggagct
     4021 atttggtatc cggcccgatg attacttgta ttccctctgc agtgagccgc tgattgaact
     4081 ctgtagctct ggagctagtg gttccctatt ctatgtgtcc agcgacgatg agttcattat
     4141 taagacagtc caacataaag aggcggaatt tctgcagaag ctgcttccag gatactacat
     4201 gaacctcaac cagaaccctc ggactttgct gcctaaattc tatggactgt actgtgtgca
     4261 ggcaggtggc aagaacattc ggattgtggt gatgaacaat cttttaccaa gatcggtaaa
     4321 aatgcatatc aaatatgacc tcaaaggctc aacctacaaa cggcgggctt cccagaaaga
     4381 gcgagagaag cctcttccca catttaaaga cctagacttc ttacaagaca tccctgatgg
     4441 tctttttttg gatgctgaca tgtacaacgc tctctgtaag accctgcagc gtgactgttt
     4501 ggtgctgcag agcttcaaga taatggatta cagcctcttg atgtcaatcc ataatataga
     4561 tcatgcacaa cgagagccct taagcagtga aacacagtac tcagttgata ctcgaagacc
     4621 ggccccccaa aaggctctgt attccacagc catggaatcc atccagggag aggctcgacg
     4681 gggtggtacc atggagactg atgaccatat gggtggcatc cctgcccgga atagtaaagg
     4741 ggaaaggctt ctgctttata ttggcatcat tgacattcta cagtcttaca ggtttgttaa
     4801 gaagttggag cactcttgga aagccctggt acatgacgga gacactgtct cagtgcatcg
     4861 cccaggcttc tacgctgaac ggttccagcg cttcatgtgc aacacagtat ttaagaagat
     4921 tcccttgaag ccttctcctt ccaaaaagtt tcggtctggc tcatctttct ctcggcgagc
     4981 aggctccagt ggcaactcct gcattactta ccagccatcg gtctctgggg aacacaaggc
     5041 acaagtgaca acaaaggcag aagtggagcc aggcgttcac cttggtcgtc ctgatgtttt
     5101 acctcagact ccacctttgg aggaaatcag tgagggctcg cctattcctg accccagttt
     5161 ctcacctcta gttggagaga ctttgcaaat gctaactaca agtacaacct tggaaaagct
     5221 tgaagttgca gagtcagagt tcacccatta acgggatcca ccggatctag ataactgatc
     5281 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5341 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5401 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5461 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     5521 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5581 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5641 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5701 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5761 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5821 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5881 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5941 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     6001 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6061 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     6121 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     6181 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     6241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     6301 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     6361 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaa
//
