LOCUS       sequence-384586-        6090 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Assessing of ubiquitination in cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6090)
  AUTHORS   Smith CE, Tsai YC, Liang YH, Khago D, Mariano J, Li J, Tarasov SG, 
            Gergel E, Tsai B, Villaneuva M, Clapp ME, Magidson V, Chari R, Byrd 
            RA, Ji X, Weissman AM
  TITLE     A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and
            is essential for ER-associated degradation.
  JOURNAL   PLoS Biol. 2021 Dec 8;19(12):e3001474. doi: 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec.
  PUBMED    34879065
REFERENCE   2  (bases 1 to 6090)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Biol."; date: "2021-12-8"; volume: "19(12):e3001474. doi"; pages: " 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec"
COMMENT     Sequence Label: pcDNA4-NHK-MYC-His6
FEATURES             Location/Qualifiers
     source          1..6090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2013..2042
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2058..2075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2098..2115)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2104..2328
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2374..2802
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2461..2480)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2671..2692
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2811..2831)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2816..3145
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2996..3131
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3058..3077
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3193..3240
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3259..3633
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3763..3884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3800..3819)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3854..3873
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3933..3949)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3933..3949)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3946..3968)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3957..3973
                     /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)."
     promoter        complement(join(3981..3987,3988..4005,4006..4011))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4026..4047
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4164..4181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4335..4923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4415..4434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5094..5885,5886..5954))
                     /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     5717..5736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5955..6059)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taaagcttgg atccgccacc atgccgtctt ctgtctcgtg gggcatcctc ctgctggcag
      961 gcctgtgctg cctggtccct gtctccctgg ctgaggatcc ccagggagat gctgcccaga
     1021 agacagatac atcccaccat gatcaggatc acccaacctt caacaagatc acccccaacc
     1081 tggctgagtt cgccttcagc ctataccgcc agctggcaca ccagtccaac agcaccaata
     1141 tcttcttctc cccagtgagc atcgctacag cctttgcaat gctctccctg gggaccaagg
     1201 ctgacactca cgatgaaatc ctggagggcc tgaatttcaa cctcacggag attccggagg
     1261 ctcagatcca tgaaggcttc caggaactcc tccgtaccct caaccagcca gacagccagc
     1321 tccagctgac caccggcaat ggcttgttcc tcagcgaggg cctgaagcta gtggataagt
     1381 ttttggagga tgttaaaaag ttgtaccact cagaagcctt cactgtcaac ttcggggaca
     1441 ccgaagaggc caagaaacag atcaacgatt acgtggagaa gggtactcaa gggaaaattg
     1501 tggatttggt caaggagctt gacagagaca cagtttttgc tctggtgaat tacatcttct
     1561 ttaaaggcaa atgggagaga ccctttgaag tcaaggacac cgaggaagag gacttccacg
     1621 tggaccaggt gaccaccgtg aaggtgccta tgatgaagcg tttaggcatg tttaacatcc
     1681 agcactgtaa gaagctgtcc agctgggtgc tgctgatgaa atacctgggc aatgccaccg
     1741 ccatcttctt cctgcctgat gaggggaaac tacagcacct ggaaaatgaa ctcacccacg
     1801 atatcatcac caagttcctg gaaaatgaag acagaaggtc tgccagctta catttaccca
     1861 aactgtccat tactggaacc tatgatctga agagcgtcct gggtcaactg ggcatcacta
     1921 aggtcttcag caatggggct gacctccggg gtcacagagg aggcacccct gaagctctcc
     1981 aaggccgtgc actcgagtct agagggccct tcgaacaaaa actcatctca gaagaggatc
     2041 tgaatatgca taccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct
     2101 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2161 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2221 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2281 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     2341 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     2401 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2461 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2521 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2581 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2641 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2701 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2761 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     2821 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2881 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2941 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3001 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3061 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3121 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3181 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     3241 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     3301 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     3361 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     3421 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     3481 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     3541 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     3601 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc
     3661 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3721 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     3781 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3841 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3901 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3961 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4021 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4081 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4141 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4201 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4261 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4321 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4381 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4441 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4501 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4561 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4621 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4681 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4741 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4801 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4861 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4921 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4981 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5041 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5101 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5161 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5221 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5281 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5341 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5401 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5461 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5521 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5581 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5641 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5701 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5761 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5821 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5881 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5941 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6001 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6061 acatttcccc gaaaagtgcc acctgacgtc
//
