LOCUS       sequence-434371-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for E. coli producing ubiquitin with 
            N-terminal YFP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  AUTHORS   Vela-Rodriguez C, Scarpulla I, Ashok Y, Lehtio L
  TITLE     Discovery of DTX3L inhibitors through a homogeneous FRET-based assay
            that monitors formation and removal of poly-ubiquitin chains.
  JOURNAL   SLAS Discov. 2023 Dec;28(8):365-375. doi: 
            10.1016/j.slasd.2023.08.005. Epub 2023 Aug 12.
  PUBMED    37579950
REFERENCE   2  (bases 1 to 6309)
  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"; doi: 
            "10.1016/j.slasd.2023.08.005"; journalName: "SLAS Discov"; date: 
            "2023-12"; volume: "28"; issue: "8"; pages: "365-375"
COMMENT     Sequence Label: Ubc-c005
FEATURES             Location/Qualifiers
     source          1..6309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(110..112,113..823)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(152..173)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(413..432)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     760..781
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             848..868
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             872..1099
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1174..1191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1244..1262)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1258..1305
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1342..1797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1429..1448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1639..1660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1889..2704)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1889..2704)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2612..2631
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2696..2715
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2826..3414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3315..3334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3568..3585
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3600..3739
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3665..3684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3825..3847
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3841..4032)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4841..5923)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5885..5904
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5924..6001)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6207..6226
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatg tgagcaaggg
      121 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      181 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      241 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
      301 cggctacggc ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
      361 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      421 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      481 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      541 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
      601 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      661 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
      721 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      781 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtcttctg gtgtagatct
      841 gggtaccgag aacttgtact tccaatccat gatgcagatc ttcgtcaaga cgttaaccgg
      901 taaaaccata actctggaag ttgaaccatc cgataccatc gaaaacgtta aggctaaaat
      961 tcaagacaag gaaggaattc cacctgatca acaaagattg atctttgccg gtaagcagct
     1021 cgaggacggt agaacgctgt ctgattacaa cattcagaag gagtcgacct tacatcttgt
     1081 cttaagacta agaggtggtt aagacggtct ccagtaaagg tggatacgga tccgaattcg
     1141 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1201 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1261 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1321 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1381 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1441 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1501 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1561 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1621 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1681 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1741 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1801 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1861 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1921 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1981 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2041 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2101 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2161 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2221 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2281 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2341 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2401 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2461 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2521 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2581 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2641 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2701 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2761 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2821 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2881 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2941 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3001 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3061 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3421 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3481 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3541 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3601 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3661 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3721 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3781 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3841 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3901 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3961 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4021 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4081 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4141 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4201 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4261 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4321 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4381 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4441 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4501 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4561 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4621 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4681 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4741 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4801 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4861 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4921 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4981 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5041 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5101 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5161 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5221 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5281 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5341 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5401 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5461 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5521 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5581 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5641 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5701 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5761 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5821 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5881 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5941 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6001 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6061 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6121 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6181 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6241 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6301 ccgcgaaat
//
