LOCUS       sequence-252411-        5061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a EGFP-human GATE-16 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5061)
  AUTHORS   Tanida I, Tanida-Miyake E, Ueno T, Kominami E
  TITLE     The human homolog of Saccharomyces cerevisiae Apg7p is a 
            Protein-activating enzyme for multiple substrates including human 
            Apg12p, GATE-16, GABARAP, and MAP-LC3.
  JOURNAL   J Biol Chem. 2001 Jan 19;276(3):1701-6. Epub 2000 Nov 28.
  PUBMED    11096062
REFERENCE   2  (bases 1 to 5061)
  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 Biol 
            Chem."; date: "2001-01-19"; volume: "276(3)"; pages: "1701-6. Epub 
            2000 Nov 28"
COMMENT     Sequence Label: pEGFP-hGATE-16
FEATURES             Location/Qualifiers
     source          1..5061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(249..251,252..254,255..965)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(249..251,252..254,255..965)
                     /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(294..315)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(555..574)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     902..923
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1485..1606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1522..1541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1576..1595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1613..2068)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1750..1771)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1962..1981
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2095..2199
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2201..2558
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2201..2392
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2224..2244)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2409..2544
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2593..3387
                     /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             2593..3387
                     /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(2647..2666)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3257..3276
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3575..3594)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3619..3666
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3995..4583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4484..4503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4758..5061
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
      601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
      661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
      721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
      781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
      901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      961 acaagtccgg actcaggtct atgaagtgga tgttcaagga ggaccactcg ctggaacaca
     1021 gatgcgtgga gtccgcgaag attcgagcga aatatcccga cagggttccg gtgattgtgg
     1081 aaaaggtctc aggctctcag attgttgaca ttgacaaacg gaagtacttg gttccatctg
     1141 atatcactgt ggctcagttc atgtggatca tcaggaaaag gatccagctt ccttctgaaa
     1201 aggcgatctt cctgtttgtg gataagacag tcccacagtc cagcctaact atgggacagc
     1261 tttacgagaa ggaaaaagat gaagatggat tcttatatgt ggcctacagc ggagagaaca
     1321 cttttggctt ctgactcgac ggtaccgcgg gcccgggatc caccggatct agataactga
     1381 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1441 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1501 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1561 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     1621 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1681 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1741 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1801 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1861 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1921 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1981 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2041 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2101 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2161 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     2221 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2281 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2341 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2401 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2461 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2521 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     2581 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2641 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2701 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2761 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2821 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2881 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2941 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3001 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3061 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3121 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3181 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3241 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3301 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3361 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3421 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3481 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3541 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     3601 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     3661 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     3721 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     3781 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     3841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4141 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4621 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4681 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
     4741 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4801 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4861 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4921 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4981 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     5041 ttagtcatcg ctattaccat g
//
