LOCUS       sequence-444149-        6216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6216)
  AUTHORS   Itakura E, Kishi C, Inoue K, Mizushima N
  TITLE     Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes 
            with mammalian Atg14 and UVRAG.
  JOURNAL   Mol Biol Cell. 2008 Dec . 19(12):5360-72.
  PUBMED    18843052
REFERENCE   2  (bases 1 to 6216)
  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: "Mol Biol 
            Cell. 2008 Dec . 19(12):5360-72."
COMMENT     Sequence Label: pEGFP-C1-hAtg14
FEATURES             Location/Qualifiers
     source          1..6216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        93..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        397..600
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     551..571
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      639..648
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(645..647,648..650,651..1361)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(645..647,648..650,651..1361)
                     /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(690..711)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(951..970)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1298..1319
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3036..3157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3073..3092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3127..3146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3164..3619)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3301..3322)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3513..3532
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3646..3750
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3752..4109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3752..3943
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3775..3795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3960..4095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4144..4938
                     /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             4144..4938
                     /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(4198..4217)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4808..4827
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5126..5145)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5170..5217
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5546..6134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6035..6054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      421 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      481 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      541 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      601 ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg agcaagggcg
      661 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      721 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      781 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
      841 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
      901 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
      961 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1021 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1081 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1141 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1201 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1261 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1321 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc agatcagctt
     1381 cgaattggaa ttccatggcg tctcccagtg ggaagggagc ccgggcgctg gaggctcctg
     1441 gctgcgggcc ccggccgctc gcccgggacc tggtggactc cgtggacgat gcggaggggc
     1501 tgtacgtggc tgtggagcgc tgcccgctgt gcaacactac ccgccggcgg ctgacctgcg
     1561 ccaaatgcgt tcagagcggc gatttcgtct acttcgacgg ccgcgaccgg gagaggttta
     1621 tcgacaagaa ggaaaggtta agccgactta agagcaagca agaagaattt cagaaagaag
     1681 tgttaaaagc tatggaagga aaatggataa cagatcagtt gagatggaaa ataatgtcct
     1741 gcaagatgag gattgaacag ttaaaacaaa caatatgtaa aggaaatgaa gaaatggaga
     1801 aaaattctga aggccttctc aaaaccaagg aaaagaatca gaagctttac agtcgagcac
     1861 aacggcacca agagaaaaag gagaagattc agaggcataa tcgcaaactt ggtgacctgg
     1921 tagaaaaaaa gaccattgac ttaagaagtc attatgagcg tctggcaaat cttcgacgat
     1981 cccatatatt agagctcacc tctgtcattt ttccaatcga ggaagtaaag acgggtgtga
     2041 gagaccccgc agatgtgtct tcagagagtg acagtgccat gacctccagc actgtgagca
     2101 agcttgctga agcccggagg acaacttacc tctcaggacg atgggtctgt gacgatcaca
     2161 acggagacac cagcattagc attacagggc cttggattag cctccctaac aatggggact
     2221 actctgccta ctacagctgg gtggaggaga agaaaacaac ccaggggcct gacatggagc
     2281 agagtaaccc tgcctacacc atcagtgctg cgctgtgcta tgcaactcag ctggtcaaca
     2341 ttctgtctca tatacttgat gtaaatcttc ccaaaaagct ctgcaacagt gaattttgtg
     2401 gcgaaaatct aagcaagcag aaatttactc gagcagtgaa gaaactgaat gcaaatattc
     2461 tttacctttg tttttctcag catgtaaatt tagatcaatt acaaccactg cataccctca
     2521 ggaatctaat gtacctggtc agtccaagct ctgaacacct aggcaggtca gggccctttg
     2581 aagtacgagc agaccttgag gagtccatgg aatttgtgga tcccggagtt gctggagaat
     2641 cagatgagag cggagatgag cgcgtcagcg atgaagaaac cgacctgggc acagactggg
     2701 agaacttgcc tagtccccgg ttttgtgata tcccttccca gtctgtggaa gtctcccaga
     2761 gtcagagcac ccaggcgtcc ccacccatcg cgagcagcag tgcaggtggg atgatctcct
     2821 ctgcagcagc ctcggtgacc tcctggttta aagcttacac tggacaccgt taagaattca
     2881 gatctcaagc ttgtcgacgg tacccgggat ccaccggatc tagataactg atcataatca
     2941 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     3001 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     3061 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3121 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat
     3181 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3241 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3301 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3361 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3421 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3481 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3541 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3601 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3661 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3721 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     3781 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3841 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3961 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4021 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4081 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     4141 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4201 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4261 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4321 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4381 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4441 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4501 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4561 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4621 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     4681 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4741 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4801 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4861 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4921 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4981 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     5041 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     5101 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     5161 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     5221 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     5281 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     5341 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     5401 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5461 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6181 cttttgctca catgttcttt cctgcgttat cccctg
//
