LOCUS       sequence-408467-        5531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of  GFP-2xPX (from SnxA)  in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5531)
  AUTHORS   Vines JH, Maib H, Buckley CM, Gueho A, Zhu Z, Soldati T, Murray DH, 
            King JS
  TITLE     A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on 
            macropinosomes and phagosomes.
  JOURNAL   J Cell Biol. 2023 Sep 4;222(9):e202209077. doi: 
            10.1083/jcb.202209077. Epub 2023 Jun 29.
  PUBMED    37382666
REFERENCE   2  (bases 1 to 5531)
  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 
            Biol."; date: "2023-09-4"; volume: "222(9):e202209077. doi"; pages: 
            " 10.1083/jcb.202209077. Epub 2023 Jun 29"
COMMENT     Sequence Label: pJSK664
FEATURES             Location/Qualifiers
     source          1..5531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2319..2440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2356..2375)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2410..2429
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2447..2902)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2584..2605)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2796..2815
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2929..3033
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3035..3392
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3035..3226
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3058..3078)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3243..3378
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3427..4221
                     /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             3427..4221
                     /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(3481..3500)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4091..4110
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4409..4428)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4453..4500
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4829..5417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5318..5337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctatggtg cagaaccgga acaacgaaat ctacatcacc
     1381 gtgcctcgga agatccaggg cgaagagggc ctgatgagaa agtacaccgc ctacgtgatc
     1441 gaggtggaag gcagcgagaa caagagatac caagtgaccc ggcggtacaa gcagttcgtg
     1501 ctgctgcata cccagctcgt cagagtgttc ggcgagcacg atctgccttc tctgcctgcc
     1561 aaagccaacg gcctgtactt cagcaaggac gaccacaccg agaagcggag agtgaacctg
     1621 caagagtacc tgcagaacct ggccaagaat cccgccatcc tgaacagccc cgtgttctac
     1681 cacttcctga agagagatga gggccagaac atcgaccacg tgccaagcag cacacccagc
     1741 catcagaatt ctgcagtcga cggtaccgcg ggcccgggat ctatggtgca gaaccggaac
     1801 aacgaaatct acatcaccgt gcctcggaag atccagggcg aagagggcct gatgagaaag
     1861 tacaccgcct acgtgatcga ggtggaaggc agcgagaaca agagatacca agtgacccgg
     1921 cggtacaagc agttcgtgct gctgcatacc cagctcgtca gagtgttcgg cgagcacgat
     1981 ctgccttctc tgcctgccaa agccaacggc ctgtacttca gcaaggacga ccacaccgag
     2041 aagcggagag tgaacctgca agagtacctg cagaacctgg ccaagaatcc cgccatcctg
     2101 aacagccccg tgttctacca cttcctgaag agagatgagg gccagaacat cgaccacgtg
     2161 ccaagcagca cacccagcca tcagaattcg gatccaccgg atctagataa ctgatcataa
     2221 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2281 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2341 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2401 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt
     2461 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2521 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2581 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2641 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2701 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2761 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2821 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     2881 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2941 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3001 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     3061 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3121 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3181 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3241 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3301 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3361 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     3421 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3481 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3541 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3601 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3661 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3721 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3781 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3841 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3901 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     3961 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4021 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4081 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4141 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4201 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4261 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4321 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4381 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     4441 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     4501 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     4561 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     4621 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     4681 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4741 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4801 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4861 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4921 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4981 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5041 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5101 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5161 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5221 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5281 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5341 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5401 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5461 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5521 ccgccatgca t
//
