LOCUS       sequence-450905-        6924 bp    DNA     circular SYN 15-APR-2025
DEFINITION  6xHis-Sumo-Ub-Ub-Ub-Ub.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  AUTHORS   Bard JAM, Bashore C, Dong KC, Martin A
  TITLE     The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize 
            Substrate Degradation.
  JOURNAL   Cell. 2019 Apr 4;177(2):286-298.e15. doi: 
            10.1016/j.cell.2019.02.031. Epub 2019 Mar 28.
  PUBMED    30929903
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: 
            "10.1016/j.cell.2019.02.031"; journalName: "Cell"; date: "2019-04-4-
            4"; volume: "177"; issue: "2"; pages: "286-298.e15"
COMMENT     Sequence Label: pAM102
FEATURES             Location/Qualifiers
     source          1..6924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..85
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     169..188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        169..187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    188..212
                     /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             227..249
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             268..285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             295..312
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             628..855
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             856..1083
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1084..1311
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1312..1539
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1593..1611)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1607..1654
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1829..1834,1835..1851,1852..1857))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1884..1902)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1970..2074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2075..2143,2144..2935)
                     /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     complement(2293..2312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3106..3694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3595..3614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3848..3865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3880..4019
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3945..3964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4105..4127
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4121..4312)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5624..6706)
                     /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     6668..6687
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6707..6784)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
       61 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
      121 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
      181 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
      241 agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg
      301 ccgcgcggca gccatatgct cgagatgagc gatcaagaag cgaaaccgag caccgaagac
      361 ctgggcgaca agaaggaagg cgaatacatc aagctgaaag ttattggtca agacagcagc
      421 gaaatccact tcaaggtgaa aatgaccacc cacctgaaga aactgaaaga gagctactgc
      481 cagcgtcaag gtgttccgat gaacagcctg cgtttcctgt ttgaaggcca gcgtatcgcg
      541 gacaaccaca ccccgaagga gctgggtatg gaggaagagg atgtgattga agtttatcag
      601 gagcaaaccg gtggccacag caccgtgatg cagatctttg ttaagaccct gaccggtaaa
      661 accattaccc tggaagtgga gagcagcgac accatcgata acgttaagag caaaatccaa
      721 gacaaggaag gcattccgcc ggatcagcaa cgtctgatct tcgcgggtaa acagctggaa
      781 gacggccgta ccctgagcga ttacaacatt caaaaggaga gcaccctgca cctggtgctg
      841 cgtctgcgtg gtggcatgca gattttcgtg aaaaccctga ccggcaaaac catcaccctg
      901 gaagtggaaa gcagcgacac cattgacaat gtgaaaagca aaatccaaga caaagagggc
      961 attccgccgg accaacagcg cctgatcttc gcgggcaagc aactggaaga tggtcgcacc
     1021 ctgagcgatt ataacattca aaaagaaagc accctgcatc tggttctgcg cctgcgtggt
     1081 ggcatgcaaa ttttcgtgaa gaccctgacc ggcaagacta ttaccctgga agtggaatct
     1141 agcgatacca ttgacaatgt gaagagcaag attcaggata aggaaggcat tccgccggac
     1201 cagcagcgcc tgatttttgc gggcaagcag ctggaagatg gtcgtaccct gagcgactat
     1261 aatatccaga aagagtctac tctgcattta gttttacgat tacgtggtgg catgcaaatc
     1321 ttcgtgaaga ctttaaccgg taagacaatt accctggaag tggagtctag cgacaccatt
     1381 gacaacgtga agagcaaaat ccaggacaaa gaaggcattc cgccggatca acagcgtctg
     1441 attttcgcgg gcaaacagct ggaggacggc cgtaccctga gcgactacaa cattcaaaag
     1501 gaaagcaccc tgcacctggt tctgcgtctg cgtggcggct aaggatccgg ctgctaacaa
     1561 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1621 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
     1681 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg
     1741 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
     1801 tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca
     1861 tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1921 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1981 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2041 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2101 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2161 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2221 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2281 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     2341 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2401 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2461 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2521 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2581 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2641 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2701 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2761 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2821 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2881 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2941 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3001 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3061 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3121 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3181 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3241 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3301 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3361 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3421 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3481 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3541 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3601 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3661 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3721 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3781 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3841 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3901 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3961 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4021 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4081 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4141 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4201 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4261 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4321 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4381 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4441 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4501 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4561 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4621 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4681 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4741 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
     4801 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt
     4861 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga
     4921 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc
     4981 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc
     5041 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga
     5101 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac
     5161 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat
     5221 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc
     5281 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5341 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5401 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5461 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5521 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5581 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5641 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5701 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5761 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5821 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5881 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     5941 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6001 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6061 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6121 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6181 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6241 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6301 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6361 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6421 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6481 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6541 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6601 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6661 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6721 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6781 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6841 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     6901 aacagtcccc cggccacggg gcct
//
