LOCUS       sequence-212611-        5192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Thermoplasma acidophilium 20S proteasome in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5192)
  AUTHORS   Yu Z, Yu Y, Wang F, Myasnikov AG, Coffino P, Cheng Y
  TITLE     Allosteric coupling between alpha-rings of the 20S proteasome.
  JOURNAL   Nat Commun. 2020 Sep 11;11(1):4580. doi: 10.1038/s41467-020-18415-7.
  PUBMED    32917864
REFERENCE   2  (bases 1 to 5192)
  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"; journalName: "Nat 
            Commun."; date: "2020-09-11"; volume: "11(1):4580. doi"; pages: " 
            10.1038/s41467-020-18415-7"
COMMENT     Sequence Label: pRSF-T20S
FEATURES             Location/Qualifiers
     source          1..5192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..750
                     /direction=RIGHT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     481..500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     723..740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        766..857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             858..1673
                     /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             858..1673
                     /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     complement(931..950)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1903..1950)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1946..1964
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2002..2046)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             2767..2789
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2804..2828
                     /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(2829..2847)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2857..2889)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(2896..2913)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2914..2934)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     RBS             3583..3605
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3620..3644
                     /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(3645..3663)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3675..3692)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3748..3772
                     /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(3773..3791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3837..3914
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3915..4997
                     /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(3934..3953)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aacggaatag ctgttcgttg acttgataga ccgattgatt catcatctca taaataaaga
       61 aaaaccaccg ctaccaacgg tggttttctc aaggttcgct gagctaccaa ctctttgaac
      121 caaggtaagt gggttggagg accgcactca ccaaaatctg ttctttcagt ttagccttaa
      181 caggtgcata acttcaagac aaagtcctct aaatcagtta ccaatggctg ctgccagtgg
      241 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      301 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      361 actgagatac caacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      421 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      481 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctggc ttgagcgtcg
      541 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgcctgcg gcgttggctt
      601 cttccggtgc tttgcttttt gctcacatgt tctttccggc tttatcccct gattctgtgg
      661 ataaccgtat taccgctttt gagtgagctg acaccgctcg ccgcagtcga acgaccgagc
      721 gtagcgagtc agtgagcgag gaagcggaag agcgctgcat gcctatttgt ttatttttct
      781 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
      841 attgaaaaag gaagagtatg agccatattc aacgggaaac gtcttgctct aggccgcgat
      901 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      961 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     1021 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1081 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     1141 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     1201 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     1261 ctgtttgtaa ttgtcctttt aacagcgacc gcgtatttcg tctcgctcag gcgcaatcac
     1321 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     1381 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     1441 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     1501 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     1561 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1621 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     1681 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1741 acatttcccc gaaaagtgcc acttgcggag acccggtcgt cagcttgtcg tcggttcagg
     1801 gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg
     1861 aagcagtgtg accgtgtgct tctcaaatgc ctgaggtttc agcaaaaaac ccctcaagac
     1921 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagcctaggt
     1981 taattaagct gcgctagtag acgagtccat gtgctggcgt tcaaatttcg cagcagcggt
     2041 ttctttacca gactcgagtc aaagaaattt tttaacttcc tcctgatcgt atatcctgta
     2101 cttatttccc accgttattg atgcgatctc cggagcctta agctcttcgc cctcctcaag
     2161 ggaagatttc agcgccttta ttccaagcgt cactgcttcc ttctcgggca ggttttcctt
     2221 gtattcgcgc tcaaggaagc tcacaacggc atctttgccc gatccgatgg ccgttgcctt
     2281 gtactcattt atagtaccgg cgggatcgca gtcgaacagc cttgggccga tctgatctat
     2341 gccagcgaag atcagcgata caccgtatgg ccttacgcca ccgtactgtg tgtactgctg
     2401 catctggtca gcaactctct taactagatt ctcaatattt acaagcgatc cgtacgttac
     2461 cttttcctgc tgggcgctta tcctggcaaa gtccacaagt actctcgcgt ctgccaccag
     2521 cccggacgtc actgcggcaa cataatcatc tataagctgt atcttttcaa ttgaattctg
     2581 ctcaataagt ctgcttctta ccttcttatc ggatatcaag agcacaccat ttgcgaattt
     2641 cataccgagc gccgttgaac ctttcttaac tgcttccctt gcatactcta cctggaaaag
     2701 tctaccatct ggtgaaaaaa ccgtaatcgc tctatcgtaa gccatctggc cctgttccat
     2761 ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
     2821 acaattcccc tatagtgagt cgtattaaag cttgcgaccc atttgctgtc caccagtcat
     2881 gctagccata tgttagtggt ggtggtggtg gtgtccctga aaatacaggt tttccgctgc
     2941 cgccagaatt aatccgagct tcctgatcct tgattctatc tgatcggtag gaagctggac
     3001 atatccgtct ttcctagtaa taaccgcaac atctatcata cctccagatg ctgaatccct
     3061 ctgctttgcc gcgcttatgg cccttatcac aagatcgaca ccttcatcga cggtcatctt
     3121 ttcgctatac tgcgattcga gaacgccgta tacgaatggt gagcctgatc ctgtgctggc
     3181 atagatatcc tcaacggaac ctccagccgc atctatggaa aatacgtggg gagctgtatc
     3241 aattccaccc acaagcagct ggaccatgta aggcatgtat tttacctggt tgagcatgtt
     3301 tgaaagcaga gtggcaacag cctctattgg catgtttact cttctctgga gcctgtaaag
     3361 ttcaagctct gccttcatat accttaccag tacttgggca tctcccacaa ggccggctat
     3421 tgtcatcccc gtataagtgt cgatctggaa cagcttttta ccattcttgt gcatgatgaa
     3481 attctccatt gtgacacgcc tttcagttgc cattattacg gcgtctttca gtgtaatacc
     3541 aaccgtggtt gtaccagttt ctaaagtctg atccatggta tatctccttc ttaaagttaa
     3601 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
     3661 ttaggatcct ggctgtggtg atgatggtga tggctgctgc ccatggtata tctccttatt
     3721 aaagttaaac aaaattattt ctacagggga attgttatcc gctcacaatt cccctatagt
     3781 gagtcgtatt aatttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     3841 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3901 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3961 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4021 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4081 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4141 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4201 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4261 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4321 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4381 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4441 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4501 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4561 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     4621 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     4681 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
     4741 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     4801 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     4861 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     4921 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4981 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     5041 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     5101 ggcatgacta acatgagaat tacaacttat atcgtatggg gctgacttca ggtgctacat
     5161 ttgaagagat aaattgcact gaaatctaga gt
//
