LOCUS       sequence-423488-        5565 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 5565)
  AUTHORS   Mizushima N, Sugita H, Yoshimori T, Ohsumi Y
  TITLE     A new protein conjugation system in human. The counterpart of the 
            yeast Apg12p conjugation system essential for autophagy.
  JOURNAL   J Biol Chem. 1998 Dec 18. 273(51):33889-92.
  PUBMED    9852036
REFERENCE   2  (bases 1 to 5565)
  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. 1998 Dec 18. 273(51):33889-92."
COMMENT     Sequence Label: pEGFP-C1-hApg5
FEATURES             Location/Qualifiers
     source          1..5565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      94..682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     583..602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        857..1160
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1161..1364
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1315..1335
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1403..1412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1409..1411,1412..1414,1415..2125)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1409..1411,1412..1414,1415..2125)
                     /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(1454..1475)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1715..1734)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2062..2083
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2973..3025
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3149..3270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3186..3205)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3240..3259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3277..3732)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3414..3435)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3626..3645
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3759..3863
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3865..4222
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3865..4056
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3888..3908)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4073..4208
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4257..5051
                     /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             4257..5051
                     /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(4311..4330)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4921..4940
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5239..5258)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5283..5330
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
       61 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      121 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      181 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      241 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      301 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      361 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      421 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      481 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      541 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      601 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      661 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      721 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      781 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      841 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      901 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      961 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     1021 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     1081 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     1141 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     1201 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     1261 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     1321 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     1381 agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1441 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1501 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1561 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1621 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1681 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1741 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1801 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1861 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1921 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1981 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2041 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2101 tctcggcatg gacgagctgt acaagtccgg actcagatcc atgacagatg acaaagatgt
     2161 gcttcgagat gtgtggtttg gacgaattcc aacttgtttc acgctatatc aggatgagat
     2221 aactgaaagg gaagcagaac catactattt gcttttgcca agagtaagtt atttgacgtt
     2281 ggtgactgac aaagtgaaaa agcactttca gaaggttatg agacaagaag acattagtga
     2341 gatatggttt gaatatgaag gcacaccact gaaatggcat tatccaattg gtttgctatt
     2401 tgatcttctt gcatcaagtt cagctcttcc ttggaacatc acagtacatt ttaagagttt
     2461 tccagaaaaa gaccttctgc actgtccatc taaggatgca attgaagctc attttatgtc
     2521 atgtatgaaa gaagctgatg ctttaaaaca taaaagtcaa gtaatcaatg aaatgcagaa
     2581 aaaagatcac aagcaactct ggatgggatt gcaaaatgac agatttgacc agttttgggc
     2641 catcaatcgg aaactcatgg aatatcctgc agaagaaaat ggatttcgtt atatcccctt
     2701 tagaatatat cagacaacga ctgaaagacc tttcattcag aagctgtttc gtcctgtggc
     2761 tgcagatgga cagttgcaca cactaggaga tctcctcaaa gaagtttgtc cttctgctat
     2821 tgatcctgaa gatggggaaa aaaagaatca agtgatgatt catggaattg agccaatgtt
     2881 ggaaacacct ctgcagtggc tgagtgaaca tctgagctac ccggataatt ttcttcatat
     2941 tagtatcatc ccacagccaa cagattgagg atctcgagct caagcttcga attctgcagt
     3001 cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     3061 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3121 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     3181 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3241 tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt
     3301 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3361 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3421 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3481 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3541 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3601 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3661 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3721 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3781 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3841 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3901 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3961 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4021 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4081 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4141 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4201 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     4261 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4321 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4381 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     4441 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4501 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4561 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4621 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4681 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4741 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4801 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4861 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4921 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4981 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5041 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5101 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5161 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5221 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     5281 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     5341 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     5401 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     5461 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     5521 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaa
//
