LOCUS       sequence-442175-        5949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacteria expression plasmid for Ubiquitin G76C with removable His6 
            tag by TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5949)
  AUTHORS   Morgan MT, Haj-Yahya M, Ringel AE, Bandi P, Brik A, Wolberger C
  TITLE     Structural basis for histone H2B deubiquitination by the SAGA DUB 
            module.
  JOURNAL   Science. 2016 Feb 12;351(6274):725-8. doi: 10.1126/science.aac5681.
  PUBMED    26912860
REFERENCE   2  (bases 1 to 5949)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2016-02-12- 12"; volume: "351"; 
            issue: "6274"; pages: "725-8"
COMMENT     Sequence Label: His6TEV-UbG76C
FEATURES             Location/Qualifiers
     source          1..5949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(331..555)
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRG"
     CDS             complement(565..585)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(607..624)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             643..665
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    680..704
                     /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     complement(704..723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(705..723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(807..826)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1032..1109
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1110..2192
                     /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     complement(1129..1148)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3504..3695
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3689..3711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3797..3936
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3852..3871
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3951..3968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4122..4710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4202..4221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4881..5672,5673..5741))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5504..5523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5742..5846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5914..5932
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcaaca accacgaagt ctcaacacaa gatgaagagt
      361 agactccttt tgaatattgt agtcagacaa agtacgtcca tcttccagct gcttgccagc
      421 aaagatcagt ctctgctgat caggaggaat tccttcctta tcctggatct tggcctttac
      481 attttctatc gtatccgagg gttcaacctc gagggtgatg gtcttccccg taagggtttt
      541 cacgaaaatc tgcatcatat ggctgccctg aaaatacagg ttttcggtgg tcggaatatc
      601 ataatcgtga tgatgatgat gatggctgct gcccatggta tatctccttc ttaaagttaa
      661 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
      721 ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
      781 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
      841 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
      901 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
      961 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1021 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1081 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1141 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1201 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1261 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1321 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1381 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1441 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1501 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1561 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1621 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1681 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1741 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1801 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     1861 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     1921 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     1981 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2041 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2101 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2161 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2221 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2281 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2341 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     2401 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     2461 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     2521 ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg
     2581 gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc
     2641 atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct
     2701 cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg
     2761 gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc
     2821 cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc
     2881 acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg
     2941 tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc
     3001 gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc
     3061 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     3121 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     3181 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     3241 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     3301 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3361 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3421 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3481 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3541 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3601 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3661 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3721 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3781 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3841 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     3901 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3961 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4021 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4081 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4141 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4201 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4261 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4321 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4381 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4441 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4501 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4561 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4621 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4681 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4741 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4801 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4861 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4921 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4981 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5041 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5101 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5161 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     5221 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5281 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5341 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5401 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5461 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     5521 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5581 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5641 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5701 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5761 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5821 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     5881 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     5941 cttcaagaa
//
