LOCUS       sequence-299751-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin with K33 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-K33
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        39..230
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(62..82)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      247..382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(416..433)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(416..432)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(425..447)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      645..1100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(732..751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     942..963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1117..1136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1236..1258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1296..1314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1382..1486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1487..1555,1556..2347)
                     /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(1705..1724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2518..3106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3007..3026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3260..3277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3394..3415
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3430..3435,3436..3453,3454..3460)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3468..3484
                     /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     3473..3495
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3492..3508
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3492..3508
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        3537..3916
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3917..4120
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4071..4091
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4117..4141
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        4351..4369
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4351..4368
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     4382..4401
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      4486..4495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4498..4524
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4555..4782
                     /codon_start=1
                     /product="human ubiquitin variant containing Lys-33, with 
                     all other lysines mutated to arginine"
                     /label=ubiquitin-K33
                     /translation="MQIFVRTLTGRTITLEVESSDTIDNVRARIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     regulatory      4819..4828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4832..4966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4869..4888)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4923..4942
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
       61 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
      181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
      241 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
      301 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
      361 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg
      421 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
      481 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
      541 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
      601 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
      661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      841 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
      901 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      961 tggaacaaca ctcaacccta tctcgggcta ttcttttaat ttataaggga ttttgccgat
     1021 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1081 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     1141 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     1201 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     1261 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     1321 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     1381 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1441 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     1501 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     1561 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     1621 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     1681 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     1741 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     1801 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     1861 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1921 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1981 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     2041 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     2101 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     2161 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     2221 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     2281 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     2341 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     2401 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     2461 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2521 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2581 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2641 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     2701 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2761 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2821 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2881 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2941 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3001 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3061 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3121 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3181 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3241 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     3301 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     3361 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     3421 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     3481 acaatttcac acaggaaaca gctatgacat gattacgaat taattcgagc tcgcccgaca
     3541 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     3601 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     3661 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     3721 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     3781 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     3841 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     3901 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     3961 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     4021 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     4081 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
     4141 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
     4201 cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag
     4261 taccgcctat agagtctata ggcccacccc cttggcttcg ttagaacgcg gctacaatta
     4321 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa taacatccac
     4381 tttgcctttc tctccacagg tgtccactcc caggtccaac tgcacctcgg ttctatcgat
     4441 tgaattcccg aaccgacagt cggtctcttc accaaggcca ttcgcgccac catgggctac
     4501 ccctatgatg tgcctgacta cgcagatctc aatggtggtg gtggtgggtc gaccatgcag
     4561 atcttcgtca gaacgttaac cggtagaacc ataactctag aagttgaatc ttccgatacc
     4621 atcgacaacg ttagagctag aattcaagac aaggaaggca ttccacctga tcaacaaaga
     4681 ttgatctttg ccggtagaca gctcgaggac ggtagaacgc tgtctgatta caacattcag
     4741 agagagtcga ccttacatct tgtcttaaga ctaagaggtg gttgagcggc cgctaagtaa
     4801 gtaaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta
     4861 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4921 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg
     4981 cgcagcacca tggcct
//
