LOCUS       Exported                4996 bp ds-DNA     circular SYN 02-SEP-2021
DEFINITION  Mammalian expression of HA tagged ubiquitin with only K48, other 
            lysines mutated to arginines.
ACCESSION   .
VERSION     .
KEYWORDS    pRK5-HA-Ubiquitin-K48
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
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Neurosci.
            2005 Feb 23. 25(8):2002-9."
FEATURES             Location/Qualifiers
     source          1..4996
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1032..1259
                     /codon_start=1
                     /product="human ubiquitin variant containing Lys-48, with 
                     all other lysines mutated to arginine"
                     /label=ubiquitin-K48
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     regulatory      1296..1305
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    1309..1443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1346..1365)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1400..1419
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1512..1869
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(1535..1555)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1720..1855
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1782..1801
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(1889..1906)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1889..1905)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1898..1920)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2118..2573
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2415..2436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     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
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2960..3820
                     /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(3178..3197)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3991..4579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4480..4499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4733..4750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4867..4888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /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
                     /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     4946..4968
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4965..4981
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4965..4981
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"