LOCUS       40924_37163        4996 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression of HA tagged ubiquitin.
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        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             1019..1246
                     /codon_start=1
                     /label=ubiquitin
                     /note="human ubiquitin C"
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1296..1430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1499..1856
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(1876..1892)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2105..2560
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2577..2596)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     2696..2718
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2756..2774)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        2842..2946
                     /label=AmpR promoter
     CDS             2947..3804
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3978..4566
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4720..4737
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4854..4875
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4890..4920
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4928..4944
                     /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     4952..4968
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgaccat
     1021 gcagatcttc gtgaagaccc tgactggtaa gaccatcact ctcgaagtgg agccgagtga
     1081 caccattgag aatgtcaagg caaagatcca agacaaggaa ggcatccctc ctgaccagca
     1141 gaggttgatc tttgctggga aacagctgga agatggacgc accctgtctg actacaacat
     1201 ccagaaagag tccaccctgc acctggtcct ccgtctcaga ggtggttgag cggccgctaa
     1261 gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca gcttataatg
     1321 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     1381 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat
     1441 tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct
     1501 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     1561 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     1621 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     1681 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     1741 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     1801 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     1861 tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1921 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1981 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     2041 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     2101 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2161 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2221 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2281 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     2341 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2401 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttaat
     2461 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2521 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     2581 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     2641 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2701 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     2761 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2821 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2881 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2941 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3001 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3061 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3121 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3181 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3241 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3301 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3361 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3421 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3481 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3541 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3601 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3661 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3721 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3781 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3841 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3901 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3961 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4021 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4081 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4141 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4201 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4261 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4321 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4381 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4441 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4501 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4561 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4621 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4681 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4741 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4801 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4861 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4921 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat
     4981 taattcgagc tcgccc
//
