LOCUS       sequence-408465-        5276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of  GFP-SnxA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5276)
  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 5276)
  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: pJSK659
FEATURES             Location/Qualifiers
     source          1..5276
                     /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    2064..2185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2101..2120)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2155..2174
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2192..2647)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2329..2350)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2541..2560
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2674..2778
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2780..3137
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2780..2971
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2803..2823)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2988..3123
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3172..3966
                     /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             3172..3966
                     /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(3226..3245)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3836..3855
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4154..4173)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4198..4245
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4574..5162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5063..5082
                     /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 atccatggaa gaactgaaca agtacaagcg ggacctcgag
     1381 aacatcagca ccaagtacat cctgctcgag aaagagaaca aagagctgcg cgagaagaac
     1441 gacgagctga accggaagct gaccatccag ctgaacaaca acgccgtgct ggaagagaag
     1501 ctgggcgtgc agaaccggaa caacgagatc tacatcaccg tgcctcggaa gatccagggc
     1561 gaagagggcc tgatgagaaa gtacaccgcc tacgtgatcg aggtggaagg cagcgagaac
     1621 aagagatacc aagtgacccg gcggtacaag cagttcgtgc tgctgcatac ccagctcgtc
     1681 agagtgttcg gcgagcacga tctgccttct ctgcctgcca aagccaacgg cctgtacttc
     1741 agcaaggacg accacaccga gaagcggaga gtgaacctgc aagagtacct gcagaacctg
     1801 gccaagaatc ccgccatcct gaacagcccc gtgttctacc acttcctgaa gagagatgag
     1861 ggccagaaca tcgaccacgt gccaagcagc acacccagcc atcgaattct gcagtcgacg
     1921 gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc cataccacat
     1981 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     2041 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2101 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2161 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     2221 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2281 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2341 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2401 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2461 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2521 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2581 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2641 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2701 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2761 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2821 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2881 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2941 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3001 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3061 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3121 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3181 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3241 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3301 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3361 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3421 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3481 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3541 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3601 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3661 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3721 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3781 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3841 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3901 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3961 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4021 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4081 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4141 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4201 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4261 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4321 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4381 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4441 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4501 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4561 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4621 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4681 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4741 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4801 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4861 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4921 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4981 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5041 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5101 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5161 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5221 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
