LOCUS       sequence-175356-        4938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Ubl4A (90-157 residues) tagged with FLAG-EGFP at N- 
            terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4938)
  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 4938)
  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-C(90-157)
FEATURES             Location/Qualifiers
     source          1..4938
                     /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    2433..2554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2470..2489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2524..2543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2561..3016)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2698..2719)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2910..2929
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3043..3147
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3149..3506
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3149..3340
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3172..3192)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3357..3492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3541..4335
                     /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             3541..4335
                     /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(3595..3614)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4205..4224
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4523..4542)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4567..4614
                     /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 gatcttcccc acccccgcag gtctggcagc
     2101 tgatctccaa agtcttggcc cgccacttca gtgcggcaga tgccagcagg gtcctggaac
     2161 agctacagag ggattacgag aggtccctga gtcgcctgac gctggacgac atcgaacggt
     2221 tggccagccg cttcctgcac cctgaagtga ctgagacaat ggagaagggc ttctccaaat
     2281 aggtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc
     2341 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2401 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2461 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2521 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2581 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2641 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2701 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2761 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2821 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2881 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2941 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3001 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3061 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3121 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3181 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3241 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3301 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3361 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3421 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3481 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3541 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3601 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3661 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3721 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3781 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3841 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3901 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3961 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4021 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4081 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4141 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4201 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4261 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4321 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4381 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4441 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4501 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4561 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4621 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4681 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4741 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4801 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4861 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4921 tagaaaagat caaaggat
//
