LOCUS       sequence-434566-        8679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8679)
  AUTHORS   Keskeny C, Raisch T, Sgromo A, Igreja C, Bhandari D, Weichenrieder 
            O, Izaurralde E
  TITLE     A conserved CAF40-binding motif in metazoan NOT4 mediates 
            association with the CCR4-NOT complex.
  JOURNAL   Genes Dev. 2019 Feb 1;33(3-4):236-252. doi: 10.1101/gad.320952.118. 
            Epub 2019 Jan 28.
  PUBMED    30692204
REFERENCE   2  (bases 1 to 8679)
  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: "Genes 
            Dev."; date: "2019-02-1"; volume: "33(3-4):236-252. doi"; pages: " 
            10.1101/gad.320952.118. Epub 2019 Jan 28"
COMMENT     Sequence Label: pnEA-NpM-HsNot4-GB1His6_AG
FEATURES             Location/Qualifiers
     source          1..8679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             88..1188
                     /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="METEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             1204..1227
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3145..3162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3181..3199)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3195..3242
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3417..3422,3423..3439,3440..3445))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3472..3490)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3558..3662
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3663..3731,3732..4523)
                     /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(3881..3900)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4694..5282
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5183..5202
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5436..5453
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5468..5607
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5533..5552)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5693..5715
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5709..5900)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     8255..8274
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8294..8371)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8577..8596
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttccccacta gtaataattt
       61 tgtttaactt taagaaggag atataccatg gaaactgaag aaggtaaact ggtaatctgg
      121 attaacggcg ataaaggcta taacggtctc gctgaagttg gtaagaaatt cgagaaagat
      181 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      241 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      301 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      361 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      421 gaagcgttat cgctgattta taacaaagac ctgctgccga acccgccaaa aacctgggaa
      481 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgccct gatgttcaac
      541 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      601 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
      661 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
      721 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
      781 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
      841 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
      901 ccgaacaaag agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
      961 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     1021 ttggcgaaag atccacgtat tgccgccacg atggaaaacg cccagaaagg tgaaatcatg
     1081 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1141 gccagcggtc gtcagactgt cgatgaagcc cttaaagacg cgcagactgg taccggcagc
     1201 ggcctggaag ttctgttcca ggggccccat atgctcgaga tgtctcgcag tcctgatgcg
     1261 aaggaagacc ctgtggagtg ccctctttgc atggagccct tggagataga tgatatcaac
     1321 tttttccctt gcacctgtgg ctaccagatt tgccgatttt gttggcatcg aattcgcact
     1381 gatgaaaatg ggctttgtcc tgcatgtaga aagccatatc cagaagaccc agcagtttat
     1441 aaaccactct cccaggaaga gctgcaaagg ataaagaatg agaaaaaaca gaaacaaaat
     1501 gagagaaaac agaaaatatc agaaaatcgc aaacatttgg ctagtgtacg tgtcgtacaa
     1561 aaaaacctcg tctttgttgt aggtttatct cagcgcctag cagacccaga ggttttaaaa
     1621 cgaccagaat attttgggaa gtttggtaaa atacataaag ttgtcatcaa taatagcaca
     1681 tcatatgcag gctcacaggg tccaagtgcc agtgcttatg taacctatat ccggtcagaa
     1741 gacgctctca gagccataca gtgtgtcaac aatgtggtag tagatggcag aacacttaag
     1801 gcatctctag gtacaacaaa atactgcagt tacttcttaa agaatatgca gtgtccaaaa
     1861 cctgactgca tgtatcttca tgaattgggg gatgaggcgg ccagcttcac aaaagaggaa
     1921 atgcaggcgg gtaaacacca agaatatgaa cagaagctac ttcaagaatt atataaatta
     1981 aatcccaatt ttcttcagct atctacgggt tcagttgata aaaataagaa caaagtgaca
     2041 ccactgcaga ggtacgatac ccccattgac aaaccttcag attctctcag tatagggaac
     2101 ggtgataatt cccagcagat atctaacagt gatacgcctt caccaccacc tggtttgtca
     2161 aaatccaatc cagtcatccc catcagttca tccaatcaca gtgcacggtc cccttttgaa
     2221 ggggcagtaa cagagtcaca gtcgttattc tcagacaatt ttcgccatcc caaccctatc
     2281 ccaagtgggc ttcctccttt ccccagctcc ccacagacat ccagtgactg gcctacagca
     2341 ccagaaccac agagcctctt cacatcagaa acaatcccag tatcatcctc tacagactgg
     2401 caagcagctt ttggctttgg ttcttctaaa caaccagagg atgacttggg ttttgatccc
     2461 ttcgatgtca ctcgaaaagc cttagcagac ctgattgaga aggaactgtc cgttcaagac
     2521 caaccttccc tttcgcccac atctcttcag aactcctctt cacacactac aaccgccaaa
     2581 ggtccaggct ctggattcct gcatcctgct gcagctacaa atgccaattc tctcaatagt
     2641 accttttcag tcttgcccca gaggttccct caatttcagc agcaccgagc ggtttataat
     2701 tcattcagtt ttccaggcca ggcagcccgc tatccttgga tggcctttcc acgcaatagc
     2761 atcatgcact tgaaccacac agcaaacccc acctcaaata gtaatttctt ggacttgaat
     2821 ctcccgccac agcacaacac aggtctggga gggatccctg tagcagggga agaagaggtg
     2881 aaggtttcga ccatgccact gtcaacctct tcccattcat tacaacaagg acagcagcct
     2941 acaagtctcc acactactgt ggccgctagc ggcatgcagt acaaacttat cctgaacggt
     3001 aaaaccctga aaggtgaaac caccaccgaa gctgttgacg ctgctaccgc ggaaaaagtt
     3061 ttcaaacagt acgctaacga caacggtgtt gacggtgaat ggacctacga cgacgctacc
     3121 aaaacctaca ccgtaacgga aggtcatcat caccatcacc actaagttgg ctgctgccac
     3181 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3241 tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac
     3301 caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt
     3361 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
     3421 aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga
     3481 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3541 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3601 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3661 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3721 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3781 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3841 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3901 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3961 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4021 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4081 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4141 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4201 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4261 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4321 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4381 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4441 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4501 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4561 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4621 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4681 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4741 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4801 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4861 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4921 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4981 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5041 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5101 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5161 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5221 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5281 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5341 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5401 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5461 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     5521 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     5581 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     5641 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5701 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5761 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5821 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     5881 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     5941 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     6001 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     6061 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     6121 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     6181 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     6241 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     6301 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     6361 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     6421 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     6481 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     6541 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     6601 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     6661 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     6721 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     6781 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     6841 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     6901 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     6961 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     7021 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     7081 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     7141 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     7201 ttgcgttgcg ctcactgccc gctttccagt cgggaaactg tcgtgccagc tgcattaatg
     7261 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     7321 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     7381 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     7441 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     7501 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     7561 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     7621 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     7681 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     7741 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     7801 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     7861 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     7921 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     7981 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     8041 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     8101 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     8161 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     8221 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     8281 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     8341 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     8401 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     8461 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     8521 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     8581 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     8641 cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
