LOCUS       sequence-472633-        7356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of VPS34 (REIE to AAAA)_FL - EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7356)
  AUTHORS   Tremel S, Ohashi Y, Morado DR, Bertram J, Perisic O, Brandt LTL, von
            Wrisberg MK, Chen ZA, Maslen SL, Kovtun O, Skehel M, Rappsilber J, 
            Lang K, Munro S, Briggs JAG, Williams RL
  TITLE     Structural basis for VPS34 kinase activation by Rab1 and Rab5 on 
            membranes.
  JOURNAL   Nat Commun. 2021 Mar 10;12(1):1564. doi: 10.1038/s41467-021-21695-2.
  PUBMED    33692360
REFERENCE   2  (bases 1 to 7356)
  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: "Nat 
            Commun."; date: "2021-03-10"; volume: "12(1):1564. doi"; pages: " 
            10.1038/s41467-021-21695-2"
COMMENT     Sequence Label: pYO1300
FEATURES             Location/Qualifiers
     source          1..7356
                     /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(3298..3300,3301..4014)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3298..3300,3301..4011)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3340..3361)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3601..3620)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3948..3969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4144..4265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4181..4200)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4235..4254
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4272..4727)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4409..4430)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4621..4640
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4754..4858
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4860..5217
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4860..5051
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4883..4903)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5068..5203
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5252..6046
                     /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             5252..6046
                     /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(5306..5325)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5916..5935
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6234..6253)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6278..6325
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /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 ccatggggga agcagagaag tttcactaca tctatagttg tgacctggat
      661 atcaacgtcc agcttaagat aggaagcttg gaagggaaga gagaacaaaa gagttataaa
      721 gctgtcctgg aagacccaat gttgaagttc tcaggactat atcaagagac atgctctgat
      781 ctttatgtta cttgtcaagt ttttgcagaa gggaagcctt tggccttgcc agtgagaaca
      841 tcctacaaag catttagtac aagatggaac tggaatgaat ggctgaaact accagtaaaa
      901 taccctgacc tgcccaggaa tgcccaagtg gccctcacca tctgggatgt gtatggtccc
      961 ggaaaagcag tgcctgtagg aggaacaacg gtttcgctct ttggaaaata cggcatgttt
     1021 cgccaaggga tgcatgactt gaaagtctgg cctaatgtag aagcagatgg atcagaaccc
     1081 acaaaaactc ctggcagaac aagtagcact ctctcagaag atcagatgag ccgtcttgcc
     1141 aagctcacca aagctcatcg acaaggacac atggtgaaag tagattggct ggatagattg
     1201 acatttgctg cagctgcaat gataaatgag agtgaaaaac gaagttctaa tttcatgtac
     1261 ctgatggttg aatttcgatg tgtcaagtgt gatgataagg aatatggtat tgtttattat
     1321 gaaaaggacg gtgatgaatc atctccaatt ttaacaagtt ttgaattagt gaaagttcct
     1381 gacccccaga tgtctatgga gaatttagtt gagagcaaac accacaagct tgcccggagt
     1441 ttaagaagtg gaccttctga ccacgatctg aaacccaatg ctgccacgag agatcagtta
     1501 aatattattg tgagttatcc accaaccaag caacttacct atgaagaaca agatcttgtt
     1561 tggaagttta gatattatct tacgaatcaa gaaaaagcct tgacaaaatt cttgaaatgt
     1621 gttaattggg atctacctca agaggccaaa caggccttgg aacttctggg aaaatggaag
     1681 ccgatggatg tagaggactc cttggagctg ttatcctctc attacaccaa cccaactgtg
     1741 aggcgttatg ctgttgcccg gttgcgacag gccgatgatg aggatttgtt gatgtaccta
     1801 ttacaattgg tccaggctct caaatatgaa aattttgatg atataaagaa tggattggaa
     1861 cctaccaaga aggatagtca gagttcagtg tcagaaaatg tgtcaaattc tggaataaat
     1921 tctgcagaaa tagatagctc ccaaattata accagccccc ttccttcagt ctcttcacct
     1981 cctcctgcat caaaaacaaa agaagttcca gatggcgaaa atctggaaca agatctctgt
     2041 accttcttga tatcgagagc ctgcaaaaac tcaacactgg ctaattattt atactggtat
     2101 gtgatagtgg aatgtgaaga tcaagatact cagcagagag atccaaagac ccatgagatg
     2161 tacttgaacg taatgagaag attcagccaa gcattgttga agggtgataa gtctgtcaga
     2221 gttatgcgtt ctttgctggc tgcacaacag acatttgtag atcggttggt gcatctaatg
     2281 aaggcagtac aacgcgaaag tggaaatcgt aagaaaaaga atgagagact acaggcattg
     2341 cttggagata atgaaaagat gaatttgtca gatgtggaac ttatcccgtt gcctttagaa
     2401 ccccaagtga aaattagagg aataattccg gaaacagcta cactgtttaa aagtgccctt
     2461 atgcctgcac agttgttttt taagacggaa gatggaggca aatatccagt tatatttaag
     2521 catggagatg atttacgtca agatcaactt attcttcaaa tcatttcact catggacaag
     2581 ctgttacgga aagaaaatct ggacttgaaa ttgacacctt ataaggtgtt agccaccagt
     2641 acaaaacatg gcttcatgca gtttatccag tcagttcctg tggctgaagt tcttgataca
     2701 gagggaagca ttcagaactt ttttagaaaa tatgcaccaa gtgagaatgg gccaaatggg
     2761 attagtgctg aggtcatgga cacttacgtt aaaagctgtg ctggatattg cgtgatcacc
     2821 tatatacttg gagttggaga caggcacctg gataaccttt tgctaacaaa aacaggcaaa
     2881 ctcttccaca tagactttgg atatattctg ggtcgtgatc caaagcctct tcctccacca
     2941 atgaagctga ataaagaaat ggtagaagga atggggggca cacagagtga gcagtaccaa
     3001 gagttccgta aacagtgtta cacggctttc ctccacctgc gaaggtattc taatctgatt
     3061 ttgaacttgt tttccttgat ggttgatgca aacattccag atattgcact tgaaccagat
     3121 aaaactgtga aaaaggttca ggataaattc cgcttagacc tgtcggatga agaggctgtg
     3181 cattacatgc agagtctgat tgatgagagt gtccatgctc tttttgctgc agtggtggaa
     3241 cagattcaca agtttgccca gtactggcgc aaaggttctg cgggatcagc tgccggagtg
     3301 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3361 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     3421 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     3481 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     3541 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     3601 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3661 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3721 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     3781 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     3841 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3901 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3961 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtgaggatcc
     4021 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     4081 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     4141 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4201 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4261 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4321 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4381 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4441 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4501 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4561 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4621 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4681 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4801 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4861 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4921 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4981 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     5041 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     5101 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     5161 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     5221 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     5281 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     5341 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     5401 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     5461 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     5521 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     5581 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     5641 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     5701 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5761 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5821 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5881 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5941 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     6001 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     6061 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     6121 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     6181 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     6241 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     6301 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     6361 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     6421 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     6481 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7321 cctgattctg tggataaccg tattaccgcc atgcat
//
