LOCUS       sequence-424404-        6653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6653)
  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 6653)
  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: pnEA-NpH-HsNot9_S
FEATURES             Location/Qualifiers
     source          1..6653
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             139..162
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1155..1173)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1169..1216
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1391..1396,1397..1413,1414..1419))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1446..1464)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1532..1636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1637..1705,1706..2497)
                     /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(1855..1874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2668..3256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3157..3176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3410..3427
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3442..3581
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3507..3526)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3667..3689
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3683..3874)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6229..6248
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6268..6345)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6551..6570
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttccccacta gtaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggtaccg gcagcggcct ggaagttctg ttccaggggc cccatatgct cgagatgcac
      181 agcctggcga cggctgcgcc tgtgcctact acactggcac aagtggatag agaaaagatc
      241 tatcagtgga tcaatgagct gtccagtcct gagactaggg aaaatgcttt gctggagcta
      301 agtaagaagc gagaatctgt tcctgacctt gcacccatgc tgtggcattc atttggtact
      361 attgcagcac ttttacagga aattgtaaat atttatccat ctatcaaccc acccaccttg
      421 acagcacacc agtctaacag agtttgcaat gctctggcat tactgcaatg tgtagcatca
      481 catccagaaa ccaggtcagc gtttctcgca gcacacatcc cacttttttt gtaccccttt
      541 ttgcacactg tcagcaaaac acgtcccttt gagtatctcc ggctcaccag ccttggagtt
      601 attggggccc tggtgaaaac agatgaacaa gaagtaatca actttttatt aacaacagaa
      661 attatccctt tatgtttgcg aattatggaa tctggaagtg aactttctaa aacagttgcc
      721 acattcatcc tccagaagat cttgttagat gacactggtt tggcttatat atgtcagacg
      781 tatgagcgtt tctcccatgt tgccatgatc ttgggtaaga tggtcctgca gctatccaaa
      841 gagccttctg cccgtctgct gaagcatgta gtgagatgtt accttcgact ttcagataac
      901 cccagggcac gtgaagcact cagacagtgc ctccctgacc agctgaaaga cacaaccttc
      961 gcccaggtgc taaaagatga caccaccacg aaacgctggc ttgcacaact ggtgaagaac
     1021 ctgcaagagg gccaggtcac cgatccccgg ggtatccccc tgccccctca gtaaggatcc
     1081 taatagtcta gagctagccc taggagatcc ggctgctaac aaagcccgaa aggaagctga
     1141 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1201 cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc
     1261 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     1321 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     1381 aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg
     1441 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     1501 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1561 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1621 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1681 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1741 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1801 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1861 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1921 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1981 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2041 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2101 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2161 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2221 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2281 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2341 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2401 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2461 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2521 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2581 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2701 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2761 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2821 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2881 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2941 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3001 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3061 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3121 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3181 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3241 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3301 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3361 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3421 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3481 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3541 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3601 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3661 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3721 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3781 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3841 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3901 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3961 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4021 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4081 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4141 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4201 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4261 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4321 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     4381 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     4441 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     4501 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     4561 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     4621 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
     4681 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     4741 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     4801 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     4861 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4921 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4981 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5041 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5101 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5161 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa actgtcgtgc
     5221 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5281 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5341 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5401 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5461 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5521 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5581 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5641 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5701 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5761 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5821 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5881 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5941 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6001 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6061 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6121 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6181 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6241 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6301 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     6361 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6421 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6481 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6541 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6601 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aat
//
