LOCUS       sequence-464046-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin with K63 only, other 
            lysines mutated to arginines.
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-K63
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     360..377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    494..515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(530..535,536..553,554..560)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    568..584
                     /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)."
     primer_bind     573..595
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     592..608
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     592..608
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        637..1016
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1017..1220
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1171..1191
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1217..1241
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1451..1469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1451..1468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1482..1501
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1586..1595
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1598..1624
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1655..1882
                     /codon_start=1
                     /product="human ubiquitin variant containing Lys-63, with 
                     all other lysines mutated to arginine"
                     /label=ubiquitin-K63
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     regulatory      1919..1928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1932..2066
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1969..1988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2023..2042
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2135..2492
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2135..2326
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2158..2178)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2343..2478
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2512..2529)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2512..2528)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2521..2543)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2741..3196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3038..3059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3213..3232)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3332..3354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3392..3410)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3478..3582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3583..3651,3652..4443)
                     /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     complement(3801..3820)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4614..4996,1..206)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
       61 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      121 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      181 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      241 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      301 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      361 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
      421 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      481 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      541 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
      601 gctatgacat gattacgaat taattcgagc tcgcccgaca ttgattattg actagttatt
      661 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      721 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      781 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      841 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      901 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      961 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     1021 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     1081 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     1141 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1201 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     1261 gctgttttga cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt
     1321 gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata
     1381 ggcccacccc cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca
     1441 tacacatacg atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg
     1501 tgtccactcc caggtccaac tgcacctcgg ttctatcgat tgaattcccg aaccgacagt
     1561 cggtctcttc accaaggcca ttcgcgccac catgggctac ccctatgatg tgcctgacta
     1621 cgcagatctc aatggtggtg gtggtgggtc gaccatgcag atcttcgtca gaacgttaac
     1681 cggtagaacc ataactctag aagttgaacc atccgatacc atcgaaaacg ttagagctag
     1741 aattcaagac agagaaggca ttccacctga tcaacaaaga ttgatctttg ccggtagaca
     1801 gctcgaggac ggtagaacgc tgtctgatta caacattcag aaggagtcca ccctgcacct
     1861 ggtcctccgt ctcagaggtg gttgagcggc cgctaagtaa gtaaggatcc ccagcttggc
     1921 cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1981 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2041 tcaatgtatc ttatcatgtc tggatcggga attaattcgg cgcagcacca tggcctgaaa
     2101 taacctctga aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga
     2161 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2221 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2281 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2341 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2401 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     2461 gaggcttttt tggaggccta ggcttttgca aaaagctgtt aacagcttgg cactggccgt
     2521 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2581 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2641 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     2701 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3061 acaacactca accctatctc gggctattct tttaatttat aagggatttt gccgatttcg
     3121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3181 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3241 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3301 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3361 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3421 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3481 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3541 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3601 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3661 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3721 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3781 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3841 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3901 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3961 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4021 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4081 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4141 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4201 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4261 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4321 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4381 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4441 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4501 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4561 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4621 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4681 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4741 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4801 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4861 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4921 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4981 gaactgagat acctac
//
