LOCUS       sequence-175665-        4788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express Ubl4A (90-107 residues) tagged with FLAG-EGFP at N- 
            terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4788)
  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 4788)
  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-H1(90-107)
FEATURES             Location/Qualifiers
     source          1..4788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      9..597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     498..517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        772..1075
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1076..1279
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1230..1250
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1318..1327
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1327..1350
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(1351..1353,1354..2064)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1351..1353,1354..2064)
                     /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(1393..1414)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1654..1673)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2001..2022
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2287..2408
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2324..2343)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2378..2397
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2415..2870)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2552..2573)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2764..2783
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2897..3001
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3003..3360
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3003..3194
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3026..3046)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3211..3346
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3395..4189
                     /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             3395..4189
                     /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(3449..3468)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4059..4078
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4377..4396)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4421..4468
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
       61 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      601 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
      661 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta
      721 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      781 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      841 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      901 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      961 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1021 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1081 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1141 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1201 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1261 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc
     1321 accatggatt acaaggatga tgatgataag gtgagcaagg gcgaggagct gttcaccggg
     1381 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1441 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1501 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1561 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1621 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1681 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1741 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1801 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1861 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1921 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1981 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2041 ctcggcatgg acgagctgta caagtccgga ctcagatctt ccccaccccc gcaggtctgg
     2101 cagctgatct ccaaagtctt ggcccgccac ttctaggtcg acggtaccgc gggcccggga
     2161 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2221 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2281 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2341 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2401 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2461 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2521 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2581 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2641 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2701 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2761 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2821 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     2881 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2941 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3001 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3061 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3121 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3181 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3241 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3301 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3361 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3421 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3481 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3541 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3601 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3661 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3721 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3781 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3841 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3901 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3961 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4021 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4081 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4141 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4201 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4261 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4321 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4381 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4441 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4501 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4561 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4621 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4681 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4741 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaa
//
