LOCUS       sequence-175353-        5001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Ubl4A (1-89 residues) tagged with FLAG-EGFP at N- 
            terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5001)
  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 5001)
  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-N(1-89)
FEATURES             Location/Qualifiers
     source          1..5001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      5..593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     494..513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        768..1071
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1072..1275
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1226..1246
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1314..1323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1323..1346
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(1347..1349,1350..2060)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1347..1349,1350..2060)
                     /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(1389..1410)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1650..1669)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1997..2018
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2496..2617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2533..2552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2587..2606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2624..3079)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2761..2782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2973..2992
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3106..3210
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3212..3569
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3212..3403
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3235..3255)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3420..3555
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3604..4398
                     /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             3604..4398
                     /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(3658..3677)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4268..4287
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4586..4605)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4630..4677
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
       61 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      121 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
      181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      661 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag
      721 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      781 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      841 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      901 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      961 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     1021 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     1081 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     1141 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     1201 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1261 tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg gtcgccacca
     1321 tggattacaa ggatgatgat gataaggtga gcaagggcga ggagctgttc accggggtgg
     1381 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1441 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1501 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1561 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1621 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1681 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1741 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1801 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1861 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1921 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1981 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2041 gcatggacga gctgtacaag tccggactca gatctatgca gctgacggtg aaggcgctgc
     2101 agggccgcga gtgcagcctg caggtgccag aggacgagct ggtgtccacg ctgaagcagc
     2161 tggtctccga gaagctgaac gtcccagtgc gccagcagcg gctgctgttc aagggcaagg
     2221 ccctggcaga tgggaaacga ctctcggatt atagcatcgg gcccaactcc aagctcaacc
     2281 tagtggtcaa acccctggag aaggtgctac tagaagaagg cgaggcccag aggctggccg
     2341 actaggtcga cggtaccgcg ggcccgggat ccaccggatc tagataactg atcataatca
     2401 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2461 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     2521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat
     2641 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2701 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2761 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2821 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2881 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2941 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3001 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3061 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3121 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3181 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     3241 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3301 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3361 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3421 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3481 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3541 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     3601 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3661 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3721 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3781 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3841 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3901 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3961 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4021 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4081 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     4141 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4201 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4261 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4321 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4381 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4441 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4501 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4561 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     4621 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     4681 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     4741 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     4801 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     4861 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4921 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4981 ccgtagaaaa gatcaaagga t
//
