LOCUS       sequence-485426-        5841 bp    DNA     circular SYN 05-SEP-2025
DEFINITION  Expresses GFP-tagged PH-Tapp1 in mammalian cells (PI(3,4)P2 
            biosensor).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5841)
  AUTHORS   Posor Y, Eichhorn-Gruenig M, Puchkov D, Schoneberg J, Ullrich A, 
            Lampe A, Muller R, Zarbakhsh S, Gulluni F, Hirsch E, Krauss M, 
            Schultz C, Schmoranzer J, Noe F, Haucke V
  TITLE     Spatiotemporal control of endocytosis by 
            phosphatidylinositol-3,4-bisphosphate.
  JOURNAL   Nature. 2013 Jul 11;499(7457):233-7. doi: 10.1038/nature12360. Epub 
            2013 Jul 3.
  PUBMED    23823722
REFERENCE   2  (bases 1 to 5841)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature12360"; journalName: "Nature"; date: "2013-07-11- 
            11"; volume: "499"; issue: "7457"; pages: "233-7"
COMMENT     Sequence Label: GFP-PH-TAPP1
FEATURES             Location/Qualifiers
     source          1..5841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        154..457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        458..661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     612..632
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      700..709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(706..708,709..711,712..1422)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(706..708,709..711,712..1422)
                     /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(751..772)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1012..1031)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1359..1380
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1438..1447
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1447..1476
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    2546..2598
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2722..2843
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2759..2778)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2813..2832
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2850..3305)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2987..3008)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3199..3218
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3332..3436
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3438..3795
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3438..3629
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3461..3481)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3646..3781
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3830..4624
                     /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             3830..4624
                     /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(3884..3903)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4494..4513
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4812..4831)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4856..4903
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5232..5820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5721..5740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
       61 gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat caattacggg
      121 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      361 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      421 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      481 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      541 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      601 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      661 tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt gagcaagggc
      721 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      781 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      841 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
      901 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
      961 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1021 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1081 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1141 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1201 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1261 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1321 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1381 accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact cagatccgcc
     1441 accatggaac aaaaactcat ctcagaagag gatctgaatg atcagcagga cctagtggaa
     1501 tgggtaaatg tgttaaacaa agctataaaa attacagtac caaagcagtc agactcacag
     1561 cctaattctg ataacctaag tcgccatggt gaatgtggga aaaagcaagt gtcttacaga
     1621 actgatattg ttggtggcgt acccatcatt actcccactc agaaagaaga agtaaatgaa
     1681 tgtggtgaaa gtattgacag aaataatctg aaacggtcac aaagccatct tccttacttt
     1741 actcctaaac cacctcaaga tagtgcggtt atcaaagctg gatattgtgt aaaacaagga
     1801 gcagtgatga aaaactggaa gagaagatat tttcaattgg atgaaaacac aataggctac
     1861 ttcaaatctg aactggaaaa ggaacctctt cgcgtaatac cacttaaaga ggttcataaa
     1921 gtccaggaat gtaagcaaag cgacataatg atgagggaca acctctttga aattgtaaca
     1981 acgtctcgaa ctttctatgt gcaggctgat agccctgaag agatgcacag ttggattaaa
     2041 gcagtctctg gcgccattgt agcacagcgg ggtcccggca gatctgcgtc ttctgagcat
     2101 ccccccggtc cttcagaatc caaacacgct ttccgtccta ccaacgcagc cgccgccacc
     2161 tcacattcca cagcctctcg cagcaactct ttggtctcaa cctttaccat ggagaagcga
     2221 ggattttacg agtctcttgc caaggtcaag ccagggaact tcaaggtcca gactgtctct
     2281 ccaagagaac cagcttccaa agtgactgaa caagctctgt taagacctca aagtaaaaat
     2341 ggccctcagg aaaaagattg tgacctagta gacttggacg atgcgagcct tccggtcagt
     2401 gacgtgtgag gcagaagcgc acggagcctg cctgcctctg ccgtcctcag ttacctttca
     2461 tgaggcttct agccaaagat gataaagggg gaaatggttt ttagtgcgta tattatactg
     2521 cctcttaggt gtactctttg aggatctcga gctcaagctt cgaattctgc agtcgacggt
     2581 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt
     2641 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2701 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2761 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2821 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt
     2881 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2941 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3001 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3061 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3121 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3181 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3241 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3301 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3361 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3421 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3481 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3541 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3601 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3661 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3721 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3781 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3841 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3901 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3961 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4021 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4081 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4141 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4201 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4261 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4321 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4381 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4441 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4501 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4561 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4621 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4681 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4741 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     4801 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     4861 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     4921 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     4981 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5041 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5101 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5161 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5221 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5281 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5341 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5401 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5461 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5521 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5581 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5641 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5701 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5761 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5821 aacgccagca acgcggcctt t
//
