LOCUS       sequence-252407-        5157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a EGFP-human Atg12/Apg12 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5157)
  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 5157)
  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-hAtg12
FEATURES             Location/Qualifiers
     source          1..5157
                     /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    1581..1702
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1618..1637)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1672..1691
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1709..2164)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1846..1867)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2058..2077
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2191..2295
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2297..2654
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2297..2488
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2320..2340)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2505..2640
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2689..3483
                     /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             2689..3483
                     /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(2743..2762)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3353..3372
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3671..3690)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3715..3762
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4091..4679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4580..4599
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4854..5157
                     /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 actcagatct ggcacgaggc cgcagtctgt gttgcagctt cctacttcaa
     1021 ttgctgctgg aggggaagga cttacggatg tctccccaga aacaaccacc ccggagcccc
     1081 cgtcttccgc tgcagtttcc ccgggaacag aggaacctgc tggcgacacc aagaaaaaaa
     1141 ttgacatttt gctaaaggct gtgggagaca ctcctattat gaaaacaaag aagtgggcag
     1201 tagagcgaac acgaaccatc caaggactca ttgacttcat caaaaagttt cttaaacttg
     1261 tggcctcaga acagttgttt atttatgtga atcagtcctt tgctccttcc ccagaccaag
     1321 aagttggaac tctctatgag tgttttggca gtgatggtaa actggtttta cattactgca
     1381 agtctcaggc gtggggatga gagctcaatc actagtgcgg ccgcctgcag gtcgacggta
     1441 ccgcgggccc gggatccacc ggatctagat aactgatcat aatcagccat accacatttg
     1501 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     1561 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     1621 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1681 ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc
     1741 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1801 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1861 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1921 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1981 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2041 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2101 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2161 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2221 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2281 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     2341 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2401 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2461 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2521 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2581 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2641 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2701 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2761 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2821 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     2881 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2941 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3001 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3061 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3121 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3181 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     3241 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3301 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3361 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3421 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3481 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3541 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3601 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     3661 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     3721 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     3781 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     3841 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     3901 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     3961 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4021 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4081 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4141 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4201 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4261 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4321 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4381 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4441 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4501 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4561 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4621 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4681 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4741 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
     4801 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     4861 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     4921 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     4981 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     5041 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     5101 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatg
//
