LOCUS       sequence-290227-        6248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot1iso1_1356-1607.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  AUTHORS   Chen Y, Boland A, Kuzuoglu-Ozturk D, Bawankar P, Loh B, Chang CT, 
            Weichenrieder O, Izaurralde E
  TITLE     A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct 
            links between miRNA target recognition and silencing.
  JOURNAL   Mol Cell. 2014 Jun 5;54(5):737-50. doi: 
            10.1016/j.molcel.2014.03.034. Epub 2014 Apr 24.
  PUBMED    24768540
REFERENCE   2  (bases 1 to 6248)
  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: 
            "10.1016/j.molcel.2014.03.034"; journalName: "Mol Cell"; date: 
            "2014-06-5- 5"; volume: "54"; issue: "5"; pages: "737-50"
COMMENT     Sequence Label: pnYC-NpM-HsNot1iso1_1356-1607_V
FEATURES             Location/Qualifiers
     source          1..6248
                     /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(4480..4498)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4494..4541
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4716..4721,4722..4738,4739..4744))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      4856..5401
                     /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     5302..5321
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5518..5535
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5927..6029
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(6030..6248,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6096..6115)
                     /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 cagtacagct accacgacat
     3661 caatgtctat tcccttgcgg gcttggcacc acacattact ctaaatccaa caattccctt
     3721 gtttcaggcc catccacagt tgaagcagtg tgtgcgtcag gcaattgaac gggctgtcca
     3781 ggagctggtc catcctgtgg tggatcgatc aattaagatt gccatgacta cttgtgagca
     3841 aatagtcagg aaggattttg ccctggattc ggaggaatct cgaatgcgaa tagcagctca
     3901 tcacatgatg cgtaacttga cagctggaat ggctatgatt acatgcaggg aacctttgct
     3961 catgagcata tctaccaact taaaaaacag ttttgcctca gcccttcgta ctgcttcccc
     4021 acaacaaaga gaaatgatgg atcaggcagc tgctcaatta gctcaggaca attgtgagtt
     4081 ggcttgctgt tttattcaga agactgcagt agaaaaagca ggccctgaga tggacaagag
     4141 attagcaact gaatttgagc tgagaaaaca tgctaggcaa gaaggacgca gatactgtga
     4201 tcctgttgtt ttaacatatc aagctgaacg gatgccagag caaatcaggc tgaaagttgg
     4261 tggtgtggac ccaaagcagt tggctgttta cgaagagttt gcacgcaatg ttcctggctt
     4321 cttacctaca aatgacttaa gtcagcccac gggattttta gcccagccca tgaagcaagc
     4381 ttgggcaaca gatgattaag gatcctaata gtctagagct agccctagga gatccggctg
     4441 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     4501 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     4561 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     4621 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     4681 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     4741 tcaaacatga gaattacaac ttatatcgta tggggctgac ttcaggtgct acatttgaag
     4801 agataaattg cactgaaatc aagaaatatt ttatctgatt aataagatga tcttcttgag
     4861 atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
     4921 gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag
     4981 cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta
     5041 actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg
     5101 ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg
     5161 tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
     5221 agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga
     5281 gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5341 ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa
     5401 aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg
     5461 aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc
     5521 gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt
     5581 gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat
     5641 agtaagccag tatacactcc cctagcgctg atgtccggcg gtgcttttgc cgttacgcac
     5701 caccccgtca gtagctgaac aggagggaca gctgatagaa acagaagcca ctggagcacc
     5761 tcaaaaacac catcatacac taaatcagta agttggcagc atcacccgac gcactttgcg
     5821 ccgaataaat acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg
     5881 ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa
     5941 gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat
     6001 cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg
     6061 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
     6121 gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa
     6181 ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc
     6241 cggaattc
//
