LOCUS       sequence-266977-        4993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin K29R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4993)
  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 4993)
  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-K29R
FEATURES             Location/Qualifiers
     source          1..4993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1293..1302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1306..1440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1343..1362)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1397..1416
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1509..1866
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1509..1700
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1532..1552)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1717..1852
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1886..1903)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1886..1902)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1895..1917)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2115..2570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2202..2221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2412..2433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2587..2606)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2706..2728
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2766..2784)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2852..2956
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2957..3025,3026..3817)
                     /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(3175..3194)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3988..4576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4477..4496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4730..4747
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4864..4885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4900..4905,4906..4923,4924..4930)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4938..4954
                     /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     4943..4965
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4962..4978
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4962..4978
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac catgcagatc ttcaagacgt taaccggtaa aaccataact ctagaagttg
     1081 aaccatccga taccatcgaa aacgttaagg ctaggattca agacaaggaa ggcattccac
     1141 ctgatcaaca aagattgatc tttgccggta agcagctcga ggacggtaga acgctgtctg
     1201 attacaacat tcagaaggag tccaccctgc acctggtcct ccgtctcaga ggtggttgag
     1261 cggccgctaa gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca
     1321 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1381 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     1441 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     1501 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     1561 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1621 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     1681 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     1741 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     1801 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     1861 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     1921 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     1981 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2041 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     2101 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2161 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2221 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2281 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     2341 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2401 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     2461 ttcttttaat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2521 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     2581 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     2641 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2701 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     2761 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2821 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2881 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2941 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3001 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3061 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3121 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3181 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3241 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3301 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3361 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3421 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3481 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3541 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3601 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3661 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3721 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3781 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3841 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3901 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3961 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4021 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4081 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4141 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4201 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4261 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4321 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4381 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4441 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4501 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4561 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4621 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4681 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4741 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     4801 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     4861 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     4921 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
     4981 gattacgaat taa
//
