LOCUS       sequence-289889-        7430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmNot10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7430)
  AUTHORS   Bawankar P, Loh B, Wohlbold L, Schmidt S, Izaurralde E
  TITLE     NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT 
            complex that docks onto the NOT1 N-terminal domain.
  JOURNAL   RNA Biol. 2013 Feb;10(2):228-44. doi: 10.4161/rna.23018. Epub 2013 
            Jan 9.
  PUBMED    23303381
REFERENCE   2  (bases 1 to 7430)
  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: "10.4161/rna.23018";
            journalName: "RNA Biol"; date: "2013-02"; volume: "10"; issue: "2"; 
            pages: "228-44"
COMMENT     Sequence Label: pnYC-NpHM-DmNot10_R
FEATURES             Location/Qualifiers
     source          1..7430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2294..2313
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2402..2421
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2402..2420
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2421..2445
                     /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             2460..2482
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2501..2518
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2534..3634
                     /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             3650..3673
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(5662..5680)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5676..5723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5898..5903,5904..5920,5921..5926))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      6038..6583
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     6484..6503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6700..6717
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        7109..7211
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(7212..7430,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(7278..7297)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ggtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
       61 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
      121 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
      181 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
      241 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
      301 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
      361 gccgtctgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
      421 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
      481 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
      541 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
      601 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
      661 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca
      721 gagcctgata aaaacggtta gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      781 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgcttg tttcggcgtg
      841 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ctgccggcac ctgtcctacg
      901 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      961 cggaaggagc taccggacag cggtgcggac tgttgtaact cagaataaga aatgaggccg
     1021 ctcatggcgt tgactctcag tcatagtatc gtggtatcac cggttggttc cactctctgt
     1081 tgcgggcaac ttcagcagca cgtaggggac ttccgcgttt ccagacttta cgaaacacgg
     1141 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     1201 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     1261 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     1321 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     1381 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     1441 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     1501 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     1561 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccagtga
     1621 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     1681 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     1741 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     1801 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     1861 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     1921 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     1981 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2041 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac
     2101 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     2161 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     2221 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     2281 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     2341 tgatgccggc cacgatgcgt ccggcgtaga ggatcagact aagatctcga tcccgcgaaa
     2401 ttaatacgac tcactatagg ggaattgtga gcggataaca attccccact agtaataatt
     2461 ttgtttaact ttaagaagga gatataccat gagcggcagc catcatcatc atcatcacag
     2521 cggcagcggc tccatggaaa ctgaagaagg taaactggta atctggatta acggcgataa
     2581 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     2641 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     2701 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     2761 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     2821 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     2881 gatttataac aaagacctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     2941 ggataaagaa ctgaaagcga aaggtaagag cgccctgatg ttcaacctgc aagaaccgta
     3001 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     3061 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     3121 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     3181 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     3241 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     3301 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     3361 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     3421 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     3481 acgtattgcc gccacgatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     3541 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     3601 gactgtcgat gaagccctta aagacgcgca gactggtacc ggcagcggcc tggaagttct
     3661 gttccagggg ccccatatgc tcgagatgga ctcggcggaa tcccccacca aaacgcaggc
     3721 cggcgaggac gagaactaca gtctgctgtg ccaggcgcac gagcagttca acaactcgga
     3781 gtttgatcgc tgcttggaac tgctccagga gttggagacg cgcggcgaga gcagtggtcc
     3841 tgtcctgcgg cacaaccggg cggtggtcag ctactacaaa accgggtgca cccaacactc
     3901 cgtcctgctg aaagaactgg aggctctaac cgccgatgca gatgcgcccg gagacgtgtc
     3961 cagcgggctg agccttaagc agggagctgc cgccgccact gttgctcgtt acaaccgggc
     4021 ggtcatctat tatcaccgcc atatgttcgg cacggcactg gagaaactag caccactagt
     4081 ggctcgtctg gaagccctgg agaaggcaat ggcagcgctg gtggccacac tacagctaca
     4141 gctgctgctg gccaccaatc aactaaaccg agccgaggcc tttctggatt acttgcagta
     4201 caagcttaac ctggtggcca cagcgcctag cagcaatgcg gccgaagagg cagctgttgt
     4261 cggcacagca cctccgtcag cagcgacgaa cagttcagtg gtggcctcac caggaggagc
     4321 agctgtggag ggagctgtgc ccggtctggg cggtagtctg cagctcttgc agctaataac
     4381 gctcgtgctt aaccgaaaac cggtggttat tcaggaagac ggcacgccgg agtacgctgc
     4441 tctgaaggct cagcagtact acatcatgaa ggattttcag atggccgcca agcagcttat
     4501 gcgaatcaat aacgagtgta cacaagcggg cactataacc ccccagctga gcacgtgcat
     4561 tgccaacaat atgggagtga tacacttaag agtgcgtcac tatgccatcg ccgccaagtt
     4621 cttccaaaat gcgctcaact ttgaccagca gctggcgcga aatttgcgcc agagtacgct
     4681 gcaaaccatg agttcggcac ggtcctgcga gatcctttac aacctcggtg tggccatgtt
     4741 gcacctacga cgccccaagg aggcgtttca gtgctttctg gttcccgtta agcaattcca
     4801 cagcaatcca cgtctttggc tgcgcatggc tgaggcctgc atcatggagc atgaggcgaa
     4861 actagtggag gaggagcgcc agtcgcagtc agaaacgcca tcaacaaagc catacgctcc
     4921 tcaatcggcg ggagttccag agcccacgct ggaattcgct gtcttgtgtc tacgtagcgc
     4981 gttaacgctt acgcagcact acaagacgag gtttcacatg gcggcggtct cctcggagga
     5041 tgtggaagca cccgaaccca aggacccaac tcaggagagt tggcgtcatc cccaggacaa
     5101 taacttctgc aatccctcga agccagtcag cctagagtct ttggaaaaca tgatggctgc
     5161 catatatgct gctcacagct tcgtctcgct gcgcctaggc gatcatgtga ccgcgctcga
     5221 gatgtcggaa aaactgctgg cctgtgaacg cttgtccgat gcacacaaat tgctaggtca
     5281 catgtacgct ggcgaggcgt tgatgcttat ggacaaggcc agcgaggcac gcgaccattt
     5341 ggaccctact tttgtgggca cactaaacgc cttcgatttt gagacgcgtg actggcaact
     5401 gaagtcggtg gacgccgctc agaacgttgt gcggtataat ctggccgttg ccatggctct
     5461 acagaacgac ctgcctcaag ctaaagcact cctggccaat ttaactcatt cgctggttgc
     5521 caacaaggcg cttgctttgc gacgcttcat ggacctcaaa atgggtcccg ttcctggcgg
     5581 gacgccgagt tagtctagag ctagccctag gagatccggc tgctaacaaa gcccgaaagg
     5641 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     5701 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     5761 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     5821 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     5881 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattaca
     5941 acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat tgcactgaaa
     6001 tcaagaaata ttttatctga ttaataagat gatcttcttg agatcgtttt ggtctgcgcg
     6061 taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct
     6121 gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg
     6181 tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta
     6241 ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag
     6301 ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg
     6361 gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa
     6421 cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc
     6481 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg
     6541 tcagatttcg tgatgcttgt caggggggcg gagcctatgg aaaaacggct ttgccgcggc
     6601 cctctcactt ccctgttaag tatcttcctg gcatcttcca ggaaatctcc gccccgttcg
     6661 taagccattt ccgctcgccg cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa
     6721 gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg
     6781 ccacatgaag cacttcactg acaccctcat cagtgccaac atagtaagcc agtatacact
     6841 cccctagcgc tgatgtccgg cggtgctttt gccgttacgc accaccccgt cagtagctga
     6901 acaggaggga cagctgatag aaacagaagc cactggagca cctcaaaaac accatcatac
     6961 actaaatcag taagttggca gcatcacccg acgcactttg cgccgaataa atacctgtga
     7021 cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc gggaagccct
     7081 gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc
     7141 ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta
     7201 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     7261 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     7321 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     7381 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc
//
