LOCUS       sequence-483541-        5425 bp    DNA     circular SYN 15-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5425)
  AUTHORS   Hosokawa N, Sasaki T, Iemura S, Natsume T, Hara T, Mizushima N
  TITLE     Atg101, a novel mammalian autophagy protein interacting with Atg13.
  JOURNAL   Autophagy. 2009 Oct . 5(7):973-9.
  PUBMED    19597335
REFERENCE   2  (bases 1 to 5425)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Autophagy. 
            2009 Oct . 5(7):973-9."
COMMENT     Sequence Label: pEGFP-C1-hAtg101
FEATURES             Location/Qualifiers
     source          1..5425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(606..608,609..611,612..1322)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(606..608,609..611,612..1322)
                     /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(651..672)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(912..931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1259..1280
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1323..1388
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2206..2327
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2243..2262)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2297..2316
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2334..2789)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2471..2492)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2683..2702
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2816..2920
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2922..3279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2922..3113
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2945..2965)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3130..3265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3314..4108
                     /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             3314..4108
                     /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(3368..3387)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3978..3997
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4296..4315)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4340..4387
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4716..5304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5205..5224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg
      601 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      661 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      721 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      781 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      841 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      901 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      961 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1021 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1081 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1141 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1201 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1261 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1321 agtccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc
     1381 cgggatccat gaactgtcgc tcggaggtgc tggaggtgtc ggtggagggg cggcaggtgg
     1441 aggaggccat gctggctgtg ctgcacacgg tgcttctgca ccgcagcaca ggcaagttcc
     1501 actacaagaa ggagggcacc tactccattg gcaccgtggg cacccaggat gttgactgtg
     1561 acttcatcga cttcacttat gtgcgtgtct cttctgagga actggatcgt gccctgcgca
     1621 aggttgttgg ggagttcaag gatgcactgc gcaactctgg tggcgatggg ctggggcaga
     1681 tgtccttgga gttctaccag aagaagaagt ctcgctggcc attctcagac gagtgcatcc
     1741 catgggaagt gtggacggtc aaggtgcatg tggtagccct ggccacggag caggagcggc
     1801 agatctgccg ggagaaggtg ggtgagaaac tctgcgagaa gatcatcaac atcgtggagg
     1861 tgatgaatcg gcatgagtac ttgcccaaga tgcccacaca gtcggaggtg gataacgtgt
     1921 ttgacacagg cttgcgggac gtgcagccct acctgtacaa gatctccttc cagatcactg
     1981 atgccctggg cacctcagtc accaccacca tgcgcaggct catcaaagac acccttgccc
     2041 tctgagcgtc gctggatctc tgggagctcc ttgatgggat ccaccggatc tagataactg
     2101 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2161 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     2221 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2281 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     2341 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2401 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2461 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2521 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2581 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2641 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2701 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2761 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     2821 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2881 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     2941 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3001 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3061 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3121 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3181 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3241 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     3301 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3361 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3421 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3481 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3541 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3601 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3661 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3721 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3781 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3841 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3901 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3961 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4021 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4081 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4141 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4201 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4261 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     4321 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     4381 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     4441 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     4501 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     4561 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4621 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4681 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4741 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4801 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4861 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4921 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4981 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5041 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5101 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5161 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5221 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5281 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5341 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5401 cgtattaccg ccatgcatta gttat
//
