LOCUS       sequence-472450-        6171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FL human EGFP-ATG14L in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6171)
  AUTHORS   Ohashi Y, Tremel S, Masson GR, McGinney L, Boulanger J, 
            Rostislavleva K, Johnson CM, Niewczas I, Clark J, Williams RL
  TITLE     Membrane characteristics tune activities of endosomal and autophagic
            human VPS34 complexes.
  JOURNAL   Elife. 2020 Jun 30;9:e58281. doi: 10.7554/eLife.58281.
  PUBMED    32602837
REFERENCE   2  (bases 1 to 6171)
  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: "Elife."; 
            date: "2020-06-30"; volume: "9:e58281. doi"; pages: " 
            10.7554/eLife.58281"
COMMENT     Sequence Label: pYO1273
FEATURES             Location/Qualifiers
     source          1..6171
                     /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    2959..3080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2996..3015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3050..3069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3087..3542)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3224..3245)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3436..3455
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3569..3673
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3675..4032
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3675..3866
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3698..3718)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3883..4018
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4067..4861
                     /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             4067..4861
                     /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(4121..4140)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4731..4750
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5049..5068)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5093..5140
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5469..6057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5958..5977
                     /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 ctgtacaagg gttctgcggg atcagctgcc ggagcgtctc ccagtgggaa gggagcccgg
     1381 gcgctggagg ctcctggctg cgggccccgg ccgctcgccc gggacctggt ggactccgtg
     1441 gacgatgcgg aggggctgta cgtggctgtg gagcgctgcc cgctgtgcaa cactacccgc
     1501 cggcggctga cctgcgccaa atgcgttcag agcggcgatt tcgtctactt cgacggccgc
     1561 gaccgggaga ggtttatcga caagaaggaa aggttaagcc gacttaagag caagcaagaa
     1621 gaatttcaga aagaagtgtt aaaagctatg gaaggaaaat ggataacaga tcagttgaga
     1681 tggaaaataa tgtcctgcaa gatgaggatt gaacagttaa aacaaacaat atgtaaagga
     1741 aatgaagaaa tggagaaaaa ttctgaaggc cttctcaaaa ccaaggaaaa gaatcagaag
     1801 ctttacagtc gagcacaacg gcaccaagag aaaaaggaga agattcagag gcataatcgc
     1861 aaacttggtg acctggtaga aaaaaagacc attgacttaa gaagtcatta tgagcgtctg
     1921 gcaaatcttc gacgatccca tatattagag ctcacctctg tcatttttcc aatcgaggaa
     1981 gtaaagacgg gtgtgagaga ccccgcagat gtgtcttcag agagtgacag tgccatgacc
     2041 tccagcactg tgagcaagct tgctgaagcc cggaggacaa cttacctctc aggacgatgg
     2101 gtctgtgacg atcacagcgg agacaccagc attagcatta cagggccttg gattagcctc
     2161 cctaacaatg gggactactc tgcctactac agctgggtgg aggagaagaa aacaacccag
     2221 gggcctgaca tggagcagag taaccctgcc tacaccatca gtgctgcgct gtgctatgca
     2281 actcagctgg tcaacattct gtctcatata cttgatgtaa atcttcccaa aaagctctgc
     2341 aacagtgaat tttgtggcga aaatctaagc aagcagaaat ttactcgagc agtgaagaaa
     2401 ctgaatgcaa atattcttta cctttgtttt tctcagcatg taaatttaga tcaattacaa
     2461 ccactgcata ccctcaggaa tctaatgtac ctggtcagtc caagctctga acacctaggc
     2521 aggtcagggc cctttgaagt acgagcagac cttgaggagt ccatggaatt tgtggatccc
     2581 ggagttgctg gagaatcaga tgagagcgga gatgagcgcg tcagcgatga agaaaccgac
     2641 ctgggcacag actgggagaa cttgcctagt ccccggtttt gtgatatccc ttcccagtct
     2701 gtggaagtct cccagagtca gagcacccag gcgtccccac ccatcgcgag cagcagtgca
     2761 ggtgggatga tctcctctgc agcagcctcg gtgacctcct ggtttaaagc ttacactgga
     2821 caccgttaag gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     2881 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     2941 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3001 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3061 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     3121 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3181 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3241 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3301 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3361 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3421 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3481 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3541 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3601 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3661 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3721 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3781 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3841 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3901 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3961 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4021 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4081 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4141 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4201 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4261 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4321 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4381 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4441 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4501 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4561 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4621 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4681 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4741 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4801 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4861 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4921 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4981 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     5041 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     5101 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     5161 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     5221 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     5281 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     5341 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5401 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5461 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5521 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5581 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5641 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5701 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5761 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5821 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5881 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5941 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6001 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6061 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6121 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
