LOCUS       sequence-235956-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin K27R and K29R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4996)
  AUTHORS   Birsa N, Norkett R, Wauer T, Mevissen TE, Wu HC, Foltynie T, Bhatia 
            K, Hirst WD, Komander D, Plun-Favreau H, Kittler JT
  TITLE     Lysine 27 ubiquitination of the mitochondrial transport protein Miro
            is dependent on serine 65 of the Parkin ubiquitin ligase.
  JOURNAL   J Biol Chem. 2014 May 23;289(21):14569-82. doi: 
            10.1074/jbc.M114.563031. Epub 2014 Mar 26.
  PUBMED    24671417
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"; doi: 
            "10.1074/jbc.M114.563031"; journalName: "J Biol Chem"; date: 
            "2014-05-23- 23"; volume: "289"; issue: "21"; pages: "14569-82"
COMMENT     Sequence Label: pRK5-HA-Ubiquitin-K27R-K29R
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        46..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..650
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        860..878
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     860..877
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     891..910
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      995..1004
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1007..1033
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1064..1291
                     /codon_start=1
                     /gene="Homo sapiens UBC"
                     /product="human ubiquitin C"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVRARIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     regulatory      1328..1337
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1341..1475
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1378..1397)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1432..1451
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1544..1901
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1544..1735
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1567..1587)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1752..1887
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1921..1938)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1921..1937)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1930..1952)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2150..2605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2237..2256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2447..2468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2622..2641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2741..2763
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2801..2819)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2887..2991
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2992..3060,3061..3852)
                     /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(3210..3229)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4023..4611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4512..4531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4765..4782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4899..4920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4935..4940,4941..4958,4959..4965)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4973..4989
                     /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     join(4978..4996,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgacatg attacgaatt aattcgagct cgcccgacat tgattattga
       61 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
      661 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc
      721 gggaacggtg cattggaacg cggattcccc gtgccaagag tgacgtaagt accgcctata
      781 gagtctatag gcccaccccc ttggcttcgt tagaacgcgg ctacaattaa tacataacct
      841 tatgtatcat acacatacga tttaggtgac actatagaat aacatccact ttgcctttct
      901 ctccacaggt gtccactccc aggtccaact gcacctcggt tctatcgatt gaattcccga
      961 accgacagtc ggtctcttca ccaaggccat tcgcgccacc atgggctacc cctatgatgt
     1021 gcctgactac gcagatctca atggtggtgg tggtgggtcg accatgcaga tcttcgtgaa
     1081 gaccctgact ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt
     1141 cagggcaagg atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc
     1201 tgggaaacag ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac
     1261 cctgcacctg gtcctccgtc tcagaggtgg ttgagcggcc gctaagtaag taaggatccc
     1321 cagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca
     1381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1441 ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat
     1501 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc
     1561 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     1621 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     1681 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     1741 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     1801 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     1861 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc
     1921 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     1981 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2041 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     2101 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     2161 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2221 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2281 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2341 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     2401 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2461 caaactggaa caacactcaa ccctatctcg ggctattctt ttaatttata agggattttg
     2521 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2581 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     2641 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2701 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     2761 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     2821 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     2881 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2941 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3001 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3061 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3121 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3181 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3241 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3301 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3361 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3421 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3481 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3541 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3601 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3661 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3721 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3781 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3841 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3901 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3961 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4021 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4081 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4141 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4201 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4261 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4321 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4381 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4441 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4501 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4561 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4621 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4681 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4741 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     4801 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4861 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     4921 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     4981 ggataacaat ttcaca
//
