LOCUS       sequence-290175-        7079 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot1iso1_1833-2361.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7079)
  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 7079)
  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: pnYC-NpM-HsNot1iso1_1833-2361_U
FEATURES             Location/Qualifiers
     source          1..7079
                     /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             2489..3589
                     /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             3605..3628
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(5311..5329)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5325..5372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5547..5552,5553..5569,5570..5575))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      5687..6232
                     /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     6133..6152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6349..6366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6758..6860
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(6861..7079,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6927..6946)
                     /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 ggaaactgaa gaaggtaaac tggtaatctg
     2521 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga
     2581 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt
     2641 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta
     2701 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta
     2761 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt
     2821 tgaagcgtta tcgctgattt ataacaaaga cctgctgccg aacccgccaa aaacctggga
     2881 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgccc tgatgttcaa
     2941 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa
     3001 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc
     3061 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta
     3121 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg
     3181 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt
     3241 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag
     3301 tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct
     3361 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga
     3421 gttggcgaaa gatccacgta ttgccgccac gatggaaaac gcccagaaag gtgaaatcat
     3481 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc
     3541 cgccagcggt cgtcagactg tcgatgaagc ccttaaagac gcgcagactg gtaccggcag
     3601 cggcctggaa gttctgttcc aggggcccca tatgctcgag cattctggga tctctcaagc
     3661 ctcagagtat gatgaccctc caggcctgag ggagaaggca gagtatcttc tgagggaatg
     3721 ggtgaatctc taccattcag cagcagctgg ccgcgacagt accaaagctt tctctgcatt
     3781 tgttggacag atgcaccagc aaggaatact gaagaccgat gatctcataa caaggttctt
     3841 tcgtctgtgt actgaaatgt gtgttgaaat cagttaccgt gctcaggctg agcagcagca
     3901 caatcctgct gccaatccca ccatgatccg agccaagtgc tatcacaacc tggatgcctt
     3961 tgttcgactc attgcactgc tcgtgaaaca ctcaggggag gccaccaaca ctgtcacaaa
     4021 gattaatctg ctgaacaagg tccttggtat agtagtggga gttctccttc aggatcatga
     4081 tgttcgtcag agtgaatttc agcaacttcc ctaccatcga atttttatca tgcttctctt
     4141 ggaactcaat gcacctgagc atgtgttgga aaccattaat ttccagacac ttacagcttt
     4201 ctgcaataca ttccacatct tgaggcctac caaagctcct ggctttgtat atgcctggct
     4261 tgaactgatt tcccatcgga tatttattgc aagaatgctg gcacatacgc cacagcagaa
     4321 ggggtggcct atgtatgcac agctactgat tgatttattc aaatatttag cgcctttcct
     4381 tagaaatgtg gaactcacca aacctatgca aatcctctac aagggcactt taagagtgct
     4441 gctggttctt ttgcatgatt tcccagagtt cctttgtgat taccattatg ggttctgtga
     4501 tgtgatccca cctaattgta tccagttaag aaatttgatc ctgagtgcct ttccaagaaa
     4561 catgaggctc cccgacccat tcactcctaa tctaaaggtg gacatgttga gtgaaattaa
     4621 cattgctccc cggattctca ccaatttcac tggagtaatg ccacctcagt tcaaaaagga
     4681 tttggattcc tatcttaaaa ctcgatcacc agtcactttc ctgtctgatc tgcgcagcaa
     4741 cctacaggta tccaatgaac ctgggaatcg ctacaacctc cagctcatca atgcactggt
     4801 gctctatgtc gggactcagg ccattgcgca catccacaac aagggcagca caccttcaat
     4861 gagcaccatc actcactcag cacacatgga tatcttccag aatttggctg tggacttgga
     4921 cactgagggt cgctatctct ttttgaatgc aattgcaaat cagctccggt acccaaatag
     4981 ccacactcac tacttcagtt gcaccatgct gtaccttttt gcagaggcca atacggaagc
     5041 catccaagaa cagatcacaa gagttctctt ggaacggttg attgtaaata ggccacatcc
     5101 ttggggtctt cttattacct tcattgagct gattaaaaac ccagcgttta agttctggaa
     5161 ccatgaattt gtacactgtg ccccagaaat cgaaaagtta ttccagtcgg tcgcacagtg
     5221 ctgcatgtaa ggatcctaat agtctagagc tagccctagg agatccggct gctaacaaag
     5281 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     5341 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
     5401 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     5461 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
     5521 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
     5581 agaattacaa cttatatcgt atggggctga cttcaggtgc tacatttgaa gagataaatt
     5641 gcactgaaat caagaaatat tttatctgat taataagatg atcttcttga gatcgttttg
     5701 gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga
     5761 aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac
     5821 caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta
     5881 aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca
     5941 agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag
     6001 tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg
     6061 cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag
     6121 ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccaccactg
     6181 atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga aaaacggctt
     6241 tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag gaaatctccg
     6301 ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg
     6361 agcgaggaag cggaatatat cctgtatcac atattctgct gacgcaccgg tgcagccttt
     6421 tttctcctgc cacatgaagc acttcactga caccctcatc agtgccaaca tagtaagcca
     6481 gtatacactc ccctagcgct gatgtccggc ggtgcttttg ccgttacgca ccaccccgtc
     6541 agtagctgaa caggagggac agctgataga aacagaagcc actggagcac ctcaaaaaca
     6601 ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc gccgaataaa
     6661 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
     6721 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
     6781 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt
     6841 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     6901 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     6961 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     7021 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattc
//
