LOCUS       sequence-313243-        6700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and protien purification of a mutated RUVBL2 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6700)
  AUTHORS   Ju D, Zhang W, Yan J, Zhao H, Li W, Wang J, Liao M, Xu Z, Wang Z, 
            Zhou G, Mei L, Hou N, Ying S, Cai T, Chen S, Xie X, Lai L, Tang C, 
            Park N, Takahashi JS, Huang N, Qi X, Zhang EE
  TITLE     Chemical perturbations reveal that RUVBL2 regulates the circadian 
            phase in mammals.
  JOURNAL   Sci Transl Med. 2020 May 6;12(542). pii: 12/542/eaba0769. doi: 
            10.1126/scitranslmed.aba0769.
  PUBMED    32376767
REFERENCE   2  (bases 1 to 6700)
  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: "Sci Transl 
            Med."; date: "2020-05-6"; volume: "12(542). pii: 12/542/eaba0769. 
            doi"; pages: " 10.1126/scitranslmed.aba0769"
COMMENT     Sequence Label: pET-28a RUVBL2 Delete
FEATURES             Location/Qualifiers
     source          1..6700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1489..1506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1516..1533
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1546..1839
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2954..2971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3024..3042)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3038..3085
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3122..3577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3209..3228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3419..3440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3669..4484)
                     /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(3669..4484)
                     /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     4392..4411
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4476..4495
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4606..5194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5095..5114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5348..5365
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5380..5519
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5445..5464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5605..5627
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5621..5812)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6621..6700,1..1003))
                     /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"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     1501 catcacagca gcggcctggt gccgcgcggc agccatatgg ctagcatgtc ggactcagaa
     1561 gtcaatcaag aagctaagcc agaggtcaag ccagaagtca agcctgagac tcacatcaat
     1621 ttaaaggtgt ccgatggatc ttcagagatc ttcttcaaga tcaaaaagac cactccttta
     1681 agaaggctga tggaagcgtt cgctaaaaga cagggtaagg aaatggactc cttaagattc
     1741 ttgtacgacg gtattaggat tcaagctgat cagacccctg aagatttgga catggaggat
     1801 aacgatatta ttgaggctca cagagaacag attggtggat ccatggcaac cgttacagcc
     1861 acaaccaaag tcccggagat ccgtgatgta acaaggattg agcgaatcgg tgcccactcc
     1921 cacatccggg gactggggct ggacgatgcc ttggagcctc ggcaggcttc gcaaggcatg
     1981 gtgggtcagc tggcggcacg gcgggcggct ggcgtggtgc tggagatgat ccgggaaggg
     2041 aagattgccg gtcgggcagt ccttattgct ggccagccgg gcacggggaa gacggccatc
     2101 gccatgggca tggcgcaggc cctgggccct gacacgccat tcacagccat cgccggcagt
     2161 gaaatcttct ccctggagat gagcaagacc gaggcgctga cgcaggcctt ccggcggtcc
     2221 atcggcgttc gcatcaagga gggtcctcca ggcgtggtgc acaccgtgtc cctgcacgag
     2281 atcgacgtca tcaactctcg cacccagggc ttcctggcgc tcttctcagg tgacacaggg
     2341 gagatcaagt cagaagtccg tgagcagatc aatgccaagg tggctgagtg gcgcgaggag
     2401 ggcaaggcgg agatcatccc tggagtgctg ttcatcgacg aggtccacat gctggacatc
     2461 gagagcttct ccttcctcaa ccgggccctg gagagtgaca tggcgcctgt cctgatcatg
     2521 gccaccaacc gtggcatcac gcgaatccgg ggcaccagct accagagccc tcacggcatc
     2581 cccatagacc tgctggaccg gctgcttatc gtctccacca ccccctacag cgagaaagac
     2641 acgaagcaga tcctccgcat ccggtgcgag gaagaagatg tggagatgag tgaggacgcc
     2701 tacacggtgc tgacccgcat cgggctggag acgtcactgc gctacgccat ccagctcatc
     2761 acagctgcca gcttggtgtg ccggaaacgc aagggtacag aagtgcaggt ggatgacatc
     2821 aagcgggtct actcactctt cctggacgag tcccgctcca cgcagtacat gaaggagtac
     2881 caggacgcct tcctcttcaa cgaactcaaa ggcgagacca tggacacctc ctaagcttgc
     2941 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     3001 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3061 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     3121 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3181 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3241 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3301 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3361 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3421 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3481 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3541 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     3601 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3661 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3721 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3781 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3841 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3901 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3961 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4021 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     4081 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     4141 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4201 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4261 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4321 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4381 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4441 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4501 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     4561 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4621 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4681 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4741 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4801 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4861 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4921 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4981 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5041 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5101 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5161 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5221 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5281 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5341 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5401 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     5461 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     5521 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5581 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5641 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     5701 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5761 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5821 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5881 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5941 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     6001 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     6061 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     6121 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     6181 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     6241 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     6301 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     6361 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     6421 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     6481 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     6541 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     6601 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6661 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
//
