LOCUS       sequence-170507-        6800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal EGFP-tagged protein expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6800)
  AUTHORS   Kim YM, Jung CH, Seo M, Kim EK, Park JM, Bae SS, Kim DH
  TITLE     mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and
            endosome maturation.
  JOURNAL   Mol Cell. 2015 Jan 22;57(2):207-18. doi: 
            10.1016/j.molcel.2014.11.013. Epub 2014 Dec 18.
  PUBMED    25533187
REFERENCE   2  (bases 1 to 6800)
  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"; doi: 
            "10.1016/j.molcel.2014.11.013"; journalName: "Mol Cell"; date: 
            "2015-01-22- 22"; volume: "57"; issue: "2"; pages: "207-18"
COMMENT     Sequence Label: EGFP-UVRAG S498D
FEATURES             Location/Qualifiers
     source          1..6800
                     /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    3588..3709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3625..3644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3679..3698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3716..4171)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3853..3874)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4065..4084
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4198..4302
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4304..4661
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4304..4495
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4327..4347)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4512..4647
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4696..5490
                     /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             4696..5490
                     /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(4750..4769)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5360..5379
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5678..5697)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5722..5769
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6098..6686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6587..6606
                     /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 actcagatct cgagtcgacc atgagcgcct ccgcgtcggt cgggggccag
     1381 gtcccccagc cacccccggg cccggccgct gctctgcctc ccggttctgc cgcgcgggcc
     1441 ctgcatgtgg agctgccgtc tcagcagcgg cgtcttcgac atcttcggaa cattgctgcc
     1501 cggaacattg ttaatagaaa tggccatcag ctccttgata cctactttac acttcacttg
     1561 tgtagtactg aaaagatata taaagaattt tatagaagtg aagtgattaa gaattccttg
     1621 aatcccacgt ggcgaagtct cgattttgga attatgccag accgtcttga tacatctgtg
     1681 tcttgtttcg tggtgaagat atggggtgga aaggagaaca tctaccagct gttgattgaa
     1741 tggaaagtct gtttggatgg gctgaaatac ttgggtcagc agattcatgc ccgaaaccaa
     1801 aatgaaataa tttttgggct gaatgatgga tactatggtg ctccatttga acataagggt
     1861 tattcaaatg ctcagaagac tattcttctg caggtggatc agaactgtgt tcgcaattct
     1921 tacgatgtct tctctttgct acggcttcat agagcccagt gtgcaattaa acagactcag
     1981 gtaactgttc agaaaattgg aaaggaaatt gaagaaaaac taagactcac atctacaagc
     2041 aatgaactga aaaaaaaaag tgaatgcctg cagttaaaaa ttttggtgct tcagaatgaa
     2101 ctggaacggc agaagaaagc tttgggacgg gaggtggcat tactgcataa gcaacaaatt
     2161 gcattacaag acaaaggaag tgcattttca gctgagcacc tcaaacttca actccagaag
     2221 gaatccctaa atgagctgag gaaggagtgc actgcaaaaa gagaactctt cttgaagact
     2281 aatgctcagt tgacaattcg ttgcaggcag ttactctctg agctttccta catttaccct
     2341 attgatttga atgaacataa ggattacttt gtatgcggtg tcaagttgcc taattctgag
     2401 gacttccaag caaaagatga tggaagcatt gctgttgccc ttggttatac tgcacatctg
     2461 gtctccatga tttccttttt cctacaagtg cccctcagat atcctataat tcataagggg
     2521 tctagatcaa caatcaaaga caatatcaat gacaaactga cggaaaagga gagagagttt
     2581 ccactgtatc caaaaggagg ggagaagttg cagtttgatt atggtgtcta tcttctgaac
     2641 aaaaatatag cacagctaag atatcaacat ggactaggga ctccagactt gcggcaaacc
     2701 cttcccaacc tgaaaaactt catggagcat ggactaatgg tcaggtgtga cagacatcac
     2761 acctccagtg caatccctgt tcctaagaga caaagctcca tatttggggg tgcagatgta
     2821 ggcttctctg gggggatccc tgacccagac aaaggacatc gaaaacgggc cagctctgag
     2881 aatgagagac ttcagtacaa aacccctcct cccagttaca actcagcatt agcccagcct
     2941 gtgaccaccg tcccctccat gggagagacc gagagaaaga taacatctct atcctcctcc
     3001 ttggatacct ccttggactt ctccaaagaa aacaagaaaa aaggagagga tctagttggc
     3061 agcttaaacg gaggccacgc gaatgtgcac cctagccaag aacaaggaga agccctctcc
     3121 gggcaccggg ccacagtcaa tggcactctc ctacccagcg agcaggccgg gtccgccagt
     3181 gtccagcttc caggcgagtt ccacccagtc tcagaagctg agctctgctg tactgtggag
     3241 caagcagaag aaatcatcgg gctggaagcc acaggtttcg cctcaggtga tcagctagaa
     3301 gcatttaact gcatcccagt ggacagtgct gtggcagtag agtgtgacga acaagttctg
     3361 ggagaatttg aagagttctc ccgaaggatc tatgcactga atgaaaacgt atccagcttc
     3421 cgccggccgc gcaggagttc cgataagtga gcggccgcag atccaccgga tctagataac
     3481 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3541 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3601 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3661 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     3721 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3781 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3841 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3901 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3961 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4021 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4081 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4141 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     4201 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4261 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     4321 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4381 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     4441 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4501 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4561 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4621 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     4681 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4741 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4801 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4861 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4921 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4981 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     5041 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     5101 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5161 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5221 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5281 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5341 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5401 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5461 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5521 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5581 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5641 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     5701 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     5761 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     5821 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     5881 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     5941 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6001 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6061 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6121 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6181 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6241 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6301 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6361 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6421 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6481 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6541 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6601 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6661 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6721 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6781 accgtattac cgccatgcat
//
