LOCUS       sequence-352283-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin K48R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4996)
  AUTHORS   Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith 
            W, Engelender S, Ross CA, Dawson VL, Dawson TM
  TITLE     Parkin mediates nonclassical, proteasomal-independent ubiquitination
            of synphilin-1: implications for Lewy body formation.
  JOURNAL   J Neurosci. 2005 Feb 23. 25(8):2002-9.
  PUBMED    15728840
REFERENCE   2  (bases 1 to 4996)
  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: "J Neurosci.
            2005 Feb 23. 25(8):2002-9."
COMMENT     Sequence Label: pRK5-HA-Ubiquitin-K48R
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..291
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     248..268
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    360..494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(384..403)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     438..457
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      498..507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(544..771)
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-48 has been 
                     mutated to arginine"
                     /label=ubiquitin-K48R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             complement(802..828)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      831..840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(925..944)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(957..975)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(958..975)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1185..1209)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1206..1409)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1235..1255)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1410..1789
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(1818..1834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1818..1834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1831..1853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1842..1858
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1866..1872,1873..1890,1891..1896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1911..1932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2049..2066)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2220..2808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2300..2319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2979..3770,3771..3839))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3602..3621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3840..3944)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4012..4030
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4068..4090)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4190..4209
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4226..4681)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4363..4384)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4575..4594
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4879..4901
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4893..4910
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4894..4910
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(4930..4996,1..291))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(4944..4996,1..83)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
       61 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
      121 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
      181 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
      241 tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca gaaggtacct
      301 aaccaagttc ctctttcaga ggttatttca ggccatggtg ctgcgccgaa ttaattcccg
      361 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
      421 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
      481 gcaataaaca agttgggcca tggcggccaa gctggggatc cttacttact tagcggccgc
      541 tcaaccacct ctgagacgga ggaccaggtg cagggtggac tccttctgaa tgttgtaatc
      601 agacagcgtt ctaccgtcct cgagctgcct accggcaaag atcaatcttt gttgatcagg
      661 tggaatgcct tccttgtctt gaattttagc cttaacgttt tcgatggtat cggatggttc
      721 aacttctaga gttatggttt taccggttaa cgtcttgacg aagatctgca tggtcgaccc
      781 accaccacca ccattgagat ctgcgtagtc aggcacatca taggggtagc ccatggtggc
      841 gcgaatggcc ttggtgaaga gaccgactgt cggttcggga attcaatcga tagaaccgag
      901 gtgcagttgg acctgggagt ggacacctgt ggagagaaag gcaaagtgga tgttattcta
      961 tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt
     1021 tctaacgaag ccaagggggt gggcctatag actctatagg cggtacttac gtcactcttg
     1081 gcacggggaa tccgcgttcc aatgcaccgt tcccggccgc ggaggctgga tcggtcccgg
     1141 tgtcttctat ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact
     1201 aaacgagctc tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat
     1261 ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc
     1321 ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc
     1381 ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg
     1441 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat
     1501 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     1561 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc
     1621 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     1681 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     1741 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtcg ggcgagctcg
     1801 aattaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2041 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2101 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2161 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2221 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2281 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2341 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2401 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2461 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2521 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2581 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2641 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2701 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2761 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2821 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2881 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2941 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3001 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3061 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3121 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3181 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3241 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3301 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3361 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3421 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3481 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3541 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3601 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3661 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3721 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3781 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3841 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3901 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3961 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4021 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4081 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4141 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4201 cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg
     4261 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4321 tataaattaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4381 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     4441 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
     4501 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4561 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4621 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4681 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     4741 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     4801 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     4861 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccaagctgt
     4921 taacagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag
     4981 ctcagaggcc gaggcg
//
