LOCUS       sequence-452269-        6924 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of yeast Ubp6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  AUTHORS   Bashore C, Dambacher CM, Goodall EA, Matyskiela ME, Lander GC, 
            Martin A
  TITLE     Ubp6 deubiquitinase controls conformational dynamics and substrate 
            degradation of the 26S proteasome.
  JOURNAL   Nat Struct Mol Biol. 2015 Sep;22(9):712-9. doi: 10.1038/nsmb.3075. 
            Epub 2015 Aug 24.
  PUBMED    26301997
REFERENCE   2  (bases 1 to 6924)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nsmb.3075";
            journalName: "Nat Struct Mol Biol"; date: "2015-09"; volume: "22"; 
            issue: "9"; pages: "712-9"
COMMENT     Sequence Label: pAM211 His-Ubp6
FEATURES             Location/Qualifiers
     source          1..6924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1476..1494
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1495..1519
                     /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)."
     CDS             1628..1672
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1710..1728)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1724..1771
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1808..2263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1895..1914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2105..2126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2378..3169,3170..3238))
                     /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     3001..3020
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3239..3331)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3412..4000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3901..3920
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4154..4171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4186..4325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4251..4270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4411..4433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4427..4618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5190..6272)
                     /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     6234..6253
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6273..6350)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6556..6575
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6663..6681
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6682..6706
                     /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             6721..6743
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6762..6779
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 aaaaatacat ggttaaaggt ggcttgtctg gcgaagagtc cattaaaata tatcccttaa
       61 tcaagccagg atcgacagta atgctattgg ggactccaga tgctaacctg atttctaaac
      121 cagccaaaaa gaataatttc attgaagacc ttgcgcctga gcaacaagtc caacaatttg
      181 ctcaattgcc tgttggtttc aagaatatgg gcaacacctg ttatctgaat gctaccctac
      241 aggctttata cagagtgaac gatttaaggg atatgattct taattataac ccttctcaag
      301 gtgtgtctaa cagtggtgca caagatgaag agattcacaa acaaatcgtt attgaaatga
      361 agcgttgttt tgaaaattta cagaataaaa gtttcaagag tgttttgcca attgtgttat
      421 taaacacgct aagaaagtgt tatccacaat ttgctgaacg tgattcacaa ggtgggttct
      481 ataaacagca agacgctgag gagttgttta cacaactatt ccatagtatg agtattgttt
      541 ttggtgacaa attttccgaa gatttcagga ttcaatttaa aactaccatc aaagacacag
      601 ctaatgataa cgatattact gttaaagaaa atgaaagcga ttctaaatta caatgtcata
      661 tttctggtac tacaaatttc atgagaaatg ggctcctgga aggtttgaat gagaaaattg
      721 aaaaaagatc agacttgact ggcgccaatt ccatctatag cgtcgaaaag aaaatatcaa
      781 gattaccaaa gtttttaact gttcagtacg ttagattttt ctggaaaagg tcaaccaaca
      841 aaaaatctaa aatattgcgt aaggtcgttt tcccatttca attagatgtt gcagacatgc
      901 ttaccccaga atacgcagca gagaaggtaa aagttcgtga cgaactgaga aaagttgaaa
      961 aggagaaaaa tgaaaaggaa agagagatca aaaggcgtaa atttgaccca tcatccagtg
     1021 aaaatgtcat gacaccaaga gaacaatatg agacacaagt ggctcttaac gaaagtgaaa
     1081 aagatcaatg gctcgaagag tataagaaac attttcctcc aaacttggaa aaaggtgaaa
     1141 acccatcttg tgtttataac ttgatcggtg tcattacaca tcaaggtgcc aattctgagt
     1201 ctggacacta tcaagctttc ataagggacg aactggacga aaataaatgg tacaaattta
     1261 atgatgataa agttagcgtt gttgaaaagg aaaaaattga atctttagcc ggtgggggcg
     1321 aaagtgatag tgcactgatc ttaatgtata aaggatttgg tctgtaagga tccgaattcg
     1381 agctcggcgc gcctgcaggt cgacaagctt gcggccgcat aatgcttaag tcgaacagaa
     1441 agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta taggggaatt
     1501 gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag gagatataca
     1561 tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg gtaccctcga
     1621 gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag
     1681 cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1741 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1801 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1861 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1921 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1981 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2041 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2101 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2161 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2221 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttctggcgg cacgatggca
     2281 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2341 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2401 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2461 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2521 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2581 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2641 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2701 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2761 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2821 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2881 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2941 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3001 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3061 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3121 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3181 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3241 tcttcctttt tcaatcatga ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3301 atatttgaat gtatttagaa aaataaacaa ataggtcatg accaaaatcc cttaacgtga
     3361 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3421 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3481 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3541 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3601 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3661 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3721 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3781 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3841 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3901 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3961 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4021 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4081 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4141 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4201 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     4261 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4321 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4381 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4441 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4501 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4561 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4621 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4681 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4741 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4801 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4861 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4921 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4981 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     5041 ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc gcccaccgga
     5101 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5161 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5221 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5281 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5341 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5401 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5461 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5521 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5581 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5641 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5701 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5761 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5821 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5881 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5941 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6001 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6061 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6121 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6181 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6241 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6301 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6361 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6421 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6481 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6541 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6601 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     6661 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     6721 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcaccat catcaccaca
     6781 gccaggatcc aatgagcgga gaaacgtttg agttcaatat tagacattct ggtaaagttt
     6841 acccaataac actttccact gatgctactt cagcagattt gaaaagcaaa gcagaggaat
     6901 tgacccaagt cccaagtgcc cgcc
//
