LOCUS       sequence-289393-        6007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot2_344-540.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6007)
  AUTHORS   Boland A, Chen Y, Raisch T, Jonas S, Kuzuoglu-Ozturk D, Wohlbold L, 
            Weichenrieder O, Izaurralde E
  TITLE     Structure and assembly of the NOT module of the human CCR4-NOT 
            complex.
  JOURNAL   Nat Struct Mol Biol. 2013 Nov;20(11):1289-97. doi: 
            10.1038/nsmb.2681. Epub 2013 Oct 13.
  PUBMED    24121232
REFERENCE   2  (bases 1 to 6007)
  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: "10.1038/nsmb.2681";
            journalName: "Nat Struct Mol Biol"; date: "2013-11"; volume: "20"; 
            issue: "11"; pages: "1289-97"
COMMENT     Sequence Label: pETM-41P-HsNot2_344-540_M
FEATURES             Location/Qualifiers
     source          1..6007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..819)
                     /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(4..819)
                     /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     727..746
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     811..830
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      941..1529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1430..1449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1683..1700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1715..1857
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1783..1802)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1943..1965
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1959..2150)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3608..3627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3711..3730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3711..3729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3761..3783
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3796..3813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3817..4917
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4891..4914
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4969..4992
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5623..5640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5693..5711)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5707..5754
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
       61 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
      121 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
      181 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
      241 gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg
      301 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
      361 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
      421 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
      481 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
      541 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
      601 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
      661 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
      721 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
      781 cggcctcgag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
      841 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
      901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
      961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1081 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     1561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     1621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     1681 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     1741 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     1801 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     1861 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1921 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1981 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     2041 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     2101 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     2161 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     2221 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     2281 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     2341 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     2401 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     2461 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     2521 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     2581 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     2641 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     2701 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     2761 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     2821 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     2881 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag
     2941 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     3001 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     3061 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     3121 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     3181 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     3241 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3301 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     3361 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgacgctctc
     3421 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     3481 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     3541 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     3601 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     3661 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     3721 cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg
     3781 agatatacca tgaaacatca ccatcaccat caccccatga aaatcgaaga aggtaaactg
     3841 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
     3901 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
     3961 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
     4021 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
     4081 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
     4141 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
     4201 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
     4261 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
     4321 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
     4381 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
     4441 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
     4501 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
     4561 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     4621 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     4681 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     4741 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
     4801 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     4861 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
     4921 tcgagctcga acaacaacaa caataacaat aacaacaacc ccatgagcct ggaagttctg
     4981 tttcagggcc ccgccatgga cggtaccgga tccactaaca ttcctcaagg gatggtgacg
     5041 gaccaatttg gaatgattgg cctgttaaca tttatcaggg cagcagagac agacccagga
     5101 atggtacatc ttgcattagg aagtgactta acaacattag gcctcaatct gaactctcct
     5161 gaaaatctct accccaaatt tgcgtcaccc tgggcatctt caccttgtcg acctcaagac
     5221 atagacttcc atgttccatc tgagtactta acgaacattc acattaggga taagctggct
     5281 gcaataaaac ttggccgata tggtgaagac cttctcttct atctctatta catgaatgga
     5341 ggagacgtat tacaactttt agctgcagtg gagcttttta accgtgattg gagataccac
     5401 aaagaagaac gagtatggat taccagggca ccaggcatgg agccaacaat gaaaaccaat
     5461 acctatgaga ggggaacata ttacttcttt gactgtctta actggaggaa agtagctaag
     5521 gagttccatc tggaatatga caaattagaa gaacggcctc acctgccatc caccttcaac
     5581 tacaaccctg ctcagcaagc cttctaagcg gccgcactcg agcaccacca ccaccaccac
     5641 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     5701 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     5761 ggaggaacta tatccggata tccacaggac gggtgtggtc gccatgatcg cgtagtcgat
     5821 agtggctcca agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc
     5881 tccgagaacg ggtgcgcata gaaattgcat caacgcatat agcgctagca gcacgccata
     5941 gtgactggcg atgctgtcgg aatggacgat atcccgcaag aggcccggca gtaccggcat
     6001 aaccaag
//
