LOCUS       40924_37158        4996 bp DNA     circular SYN 02-SEP-2021
DEFINITION  Mammalian expression of HA tagged ubiquitin with only K48, 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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4996)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Neurosci.
            2005 Feb 23. 25(8):2002-9."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4996
                     /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     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     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
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             1032..1259
                     /codon_start=1
                     /label=ubiquitin-K48
                     /note="human ubiquitin variant containing Lys-48, with all
                     other lysines mutated to arginine"
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     regulatory      1296..1305
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1309..1443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1512..1869
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(1889..1905)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2118..2573
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2590..2609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     2709..2731
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2769..2787)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        2855..2959
                     /label=AmpR promoter
     CDS             2960..3817
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3991..4579
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4733..4750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4867..4888
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4903..4933
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4941..4957
                     /label=lac operator
                     /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     4965..4981
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 ttcgtcagaa cgttaaccgg tagaaccata actctagaag
     1081 ttgaaccatc cgataccatc gaaaacgtta gagctagaat tcaagacaga gaaggcattc
     1141 cacctgatca acaaagattg atctttgccg gtaagcagct cgaggacggt agaacgctgt
     1201 ctgattacaa cattcagaga gagtccaccc tgcacctggt cctccgtctc agaggtggtt
     1261 gagcggccgc taagtaagta aggatcccca gcttggccgc catggcccaa cttgtttatt
     1321 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     1381 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     1441 atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
     1501 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     1561 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     1621 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1681 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     1741 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     1801 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     1861 ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt cgtgactggg
     1921 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     1981 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2041 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2101 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2161 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2221 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2281 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2341 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2401 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     2461 ctattctttt aatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2521 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     2581 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2641 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2701 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     2761 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2821 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2881 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2941 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3001 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3061 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3121 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3181 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3241 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3301 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3361 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3421 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3481 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3541 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3601 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3661 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3721 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3781 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3841 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3901 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3961 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4021 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4081 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4141 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4201 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4261 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4321 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4381 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4441 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4501 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4561 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4621 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4681 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4741 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     4801 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4861 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4921 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     4981 catgattacg aattaa
//
