LOCUS       sequence-177122-        4959 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 4959)
  AUTHORS   Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG
  TITLE     Short-lived green fluorescent proteins for quantifying 
            ubiquitin/proteasome-dependent proteolysis in living cells.
  JOURNAL   Nat Biotechnol. 2000 May . 18(5):538-43.
  PUBMED    10802622
REFERENCE   2  (bases 1 to 4959)
  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: "Nat 
            Biotechnol. 2000 May . 18(5):538-43."
COMMENT     Sequence Label: Ub-G76V-GFP
FEATURES             Location/Qualifiers
     source          1..4959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        423..626
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     577..597
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             696..920
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRG"
     regulatory      957..966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(963..965,966..968,969..1682)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(963..965,966..968,969..1679)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1008..1029)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1269..1288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1616..1637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1805..1926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1842..1861)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1896..1915
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1933..2388)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2070..2091)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2282..2301
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2415..2519
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2521..2878
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2521..2712
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2544..2564)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2729..2864
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2913..3707
                     /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             2913..3707
                     /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(2967..2986)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3577..3596
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3895..3914)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3939..3986
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4315..4903
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4804..4823
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
       61 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      481 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      541 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      601 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      661 ggactcagat ctcgagctca agcttcgaat tcaccatgca gatcttcgtg aagactctga
      721 ctggtaagac catcaccctc gaggttgagc ccagtgacac cattgagaat gtcaaggcaa
      781 agatccaaga taaggaaggc atccctcctg accagcagag gctgatcttt gctggaaaac
      841 agctggaaga tgggcgcacc ctgtctgact acaacatcca gaaagagtcc accctgcacc
      901 tggtactccg tctcagaggt gtggtgggga agcttggtcg acaggatcca ccggtcgcca
      961 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1021 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1081 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1141 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1201 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1261 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1321 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1381 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1441 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1741 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1801 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1861 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1921 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     1981 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2041 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2101 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2161 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2221 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2281 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2341 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2401 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2461 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2521 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2581 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2641 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2701 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2761 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2821 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     2881 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     2941 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3001 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3061 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3121 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3181 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3241 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3301 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3361 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3421 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3481 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3541 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3601 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3661 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3721 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3781 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3841 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     3901 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     3961 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4021 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4081 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4141 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4201 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4261 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4321 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4381 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4441 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4501 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4561 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4621 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4681 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4741 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4801 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4861 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4921 ctttttacgg ttcctggcct tttgctggcc ttttgctca
//
