LOCUS       sequence-175328-        5205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Ubl4A tagged with FLAG-EGFP at N- terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5205)
  AUTHORS   Xu Y, Anderson DE, Ye Y
  TITLE     The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble 
            proteins for degradation.
  JOURNAL   Cell Discov. 2016 Nov 8;2:16040. eCollection 2016.
  PUBMED    27867533
REFERENCE   2  (bases 1 to 5205)
  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: "Cell 
            Discov."; date: "2016-11-8"; volume: "2"; pages: "16040. eCollection
            2016"
COMMENT     Sequence Label: pEGFP-C1-FLAG-GFP-Ubl4A
FEATURES             Location/Qualifiers
     source          1..5205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     500..519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        774..1077
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1078..1281
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1232..1252
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1320..1329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1329..1352
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(1353..1355,1356..2066)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1353..1355,1356..2066)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1395..1416)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1656..1675)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2003..2024
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2706..2827
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2743..2762)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2797..2816
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2834..3289)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2971..2992)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3183..3202
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3316..3420
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3422..3779
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3422..3613
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3445..3465)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3630..3765
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3814..4608
                     /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             3814..4608
                     /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(3868..3887)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4478..4497
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4796..4815)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4840..4887
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
       61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      121 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
      181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      661 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
      721 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      781 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      841 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      901 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      961 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1021 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     1081 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     1141 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1201 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1261 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg
     1321 ccaccatgga ttacaaggat gatgatgata aggtgagcaa gggcgaggag ctgttcaccg
     1381 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1441 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1501 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1561 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1621 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1681 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1741 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1801 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1861 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1921 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1981 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2041 ctctcggcat ggacgagctg tacaagtccg gactcagatc tatgcagctg acggtgaagg
     2101 cgctgcaggg ccgcgagtgc agcctgcagg tgccagagga cgagctggtg tccacgctga
     2161 agcagctggt ctccgagaag ctgaacgtcc cagtgcgcca gcagcggctg ctgttcaagg
     2221 gcaaggccct ggcagatggg aaacgactct cggattatag catcgggccc aactccaagc
     2281 tcaacctagt ggtcaaaccc ctggagaagg tgctactaga agaaggcgag gcccagaggc
     2341 tggccgactc cccacccccg caggtctggc agctgatctc caaagtcttg gcccgccact
     2401 tcagtgcggc agatgccagc agggtcctgg aacagctaca gagggattac gagaggtccc
     2461 tgagtcgcct gacgctggac gacatcgaac ggttggccag ccgcttcctg caccctgaag
     2521 tgactgagac aatggagaag ggcttctcca aataggtcga cggtaccgcg ggcccgggat
     2581 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     2641 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2701 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2761 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2821 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2881 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2941 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3001 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3061 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3121 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3181 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3241 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     3301 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3361 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3421 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3481 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3541 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3601 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3661 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3721 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3781 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3841 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3901 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3961 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     4021 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4081 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4141 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4201 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4261 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4321 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     4381 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4441 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4501 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4561 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4621 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4681 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4741 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4801 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4861 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     4921 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     4981 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     5041 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     5101 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5161 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatca
//
