LOCUS       sequence-449431-        6221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Ub-G76V-GFP along dsRED from one mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6221)
  AUTHORS   Yu ZQ, Carmichael J, Collins GA, D'Agostino MD, Lessard M, Firth HV,
            Harijan P, Fry AE, Dean J, Zhang J, Kini U, Goldberg AL, Rubinsztein
            DC
  TITLE     PSMC5 insufficiency and P320R mutation impair proteasome function.
  JOURNAL   Hum Mol Genet. 2024 May 22:ddae085. doi: 10.1093/hmg/ddae085.
  PUBMED    38776958
REFERENCE   2  (bases 1 to 6221)
  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: "Hum Mol 
            Genet. 2024 May 22:ddae085. doi: 10.1093/hmg/ddae085."
COMMENT     Sequence Label: pUb-G76V-GFP-IRES-DsRed
FEATURES             Location/Qualifiers
     source          1..6221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1333..1557
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRG"
     regulatory      1594..1603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1600..1602,1603..1605,1606..2319)
                     /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(1600..1602,1603..1605,1606..2316)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1645..1666)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1906..1925)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2253..2274
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(2326..2900,2901..2903,2904..2912)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2493..2510)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2720..2739
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2913..3590
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3093..3113)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3311..3330
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3505..3528
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3712..3833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3749..3768)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3803..3822
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3840..4295)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3977..3998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4189..4208
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4322..4426
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4428..4785
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4428..4619
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4451..4471)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4636..4771
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4820..5614
                     /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             4820..5614
                     /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(4874..4893)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5484..5503
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5802..5821)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5846..5893
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     1321 gctcaagctt cgatgcagat cttcgtgaag actctgactg gtaagaccat caccctcgag
     1381 gttgagccca gtgacaccat tgagaatgtc aaggcaaaga tccaagataa ggaaggcatc
     1441 cctcctgacc agcagaggct gatctttgct ggaaaacagc tggaagatgg gcgcaccctg
     1501 tctgactaca acatccagaa agagtccacc ctgcacctgg tactccgtct cagaggtgtg
     1561 gtggggaagc ttggtcgaca ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     1621 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1681 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1741 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1801 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1861 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1921 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1981 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2041 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2101 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2161 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2221 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2281 gccgggatca ctctcggcat ggacgagctg tacaagtaag atccgcccct ctccctcccc
     2341 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat
     2401 gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
     2461 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt
     2521 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc
     2581 gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc
     2641 acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat
     2701 agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc
     2761 ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg
     2821 tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
     2881 ttgaaaaaca cgatgataat atggccacaa ccatggatag cactgagaac gtcatcaagc
     2941 ccttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     3001 agggcgaggg cgagggcaag ccctacgagg gcacccagac cgccaagctg caggtgacca
     3061 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca
     3121 aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg
     3181 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     3241 actcctccct gcaggacggc accttcatct accacgtgaa gttcatcggc gtgaacttcc
     3301 cctccgacgg ccccgtaatg cagaagaaga ctctgggctg ggagccctcc accgagcgcc
     3361 tgtacccccg cgacggcgtg ctgaagggcg agatccacaa ggcgctgaag ctgaagggcg
     3421 gcggccacta cctggtggag ttcaagtcaa tctacatggc caagaagccc gtgaagctgc
     3481 ccggctacta ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacaccg
     3541 tggtggagca gtacgagcgc gccgaggccc gccaccacct gttccagtag gcggccgcga
     3601 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3661 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     3721 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3781 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     3841 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3901 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3961 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     4021 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     4081 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     4141 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     4201 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     4261 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     4321 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4381 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     4441 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     4501 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     4561 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     4621 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4681 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4741 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     4801 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4861 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4921 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4981 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     5041 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     5101 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     5161 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     5221 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     5281 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     5341 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5401 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5461 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5521 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5581 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5641 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5701 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5761 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     5821 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     5881 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     5941 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     6001 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     6061 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6121 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6181 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
