LOCUS       sequence-291189-        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6344)
  AUTHORS   Sgromo A, Raisch T, Backhaus C, Keskeny C, Alva V, Weichenrieder O, 
            Izaurralde E
  TITLE     Drosophila Bag-of-marbles directly interacts with the CAF40 subunit 
            of the CCR4-NOT complex to elicit repression of mRNA targets.
  JOURNAL   RNA. 2018 Mar;24(3):381-395. doi: 10.1261/rna.064584.117. Epub 2017 
            Dec 18.
  PUBMED    29255063
REFERENCE   2  (bases 1 to 6344)
  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: 
            "10.1261/rna.064584.117"; journalName: "RNA"; date: "2018-03"; 
            volume: "24"; issue: "3"; pages: "381-395"
COMMENT     Sequence Label: pnYC-NpM-HsNot7_AF
FEATURES             Location/Qualifiers
     source          1..6344
                     /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(4576..4594)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4590..4637
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4812..4817,4818..4834,4835..4840))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      4952..5497
                     /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     5398..5417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5614..5631
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6023..6125
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(6126..6344,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6192..6211)
                     /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 ccagcggcaa ctgtagatca
     3661 tagccaaaga atttgtgaag tttgggcttg caacttggat gaagagatga agaaaattcg
     3721 tcaagttatc cgaaaatata attacgttgc tatggacacc gagtttccag gtgtggttgc
     3781 aagacccatt ggagaattca ggagcaatgc tgactatcaa taccaactat tgcggtgtaa
     3841 tgtagacttg ttaaagataa ttcagctagg actgacattt atgaatgagc aaggagaata
     3901 ccctccagga acttcaactt ggcagtttaa ttttaaattt aatttgacgg aggacatgta
     3961 tgcccaggac tctatagagc tactaacaac atctggtatc cagtttaaaa aacatgagga
     4021 ggaaggaatt gaaacccagt actttgcaga acttcttatg acttctggag tggtcctctg
     4081 tgaaggggtc aaatggttgt catttcatag cggttacgac tttggctact taatcaaaat
     4141 cctaaccaac tctaacttgc ctgaagaaga acttgacttc tttgagatcc ttcgattgtt
     4201 ttttcctgtc atttatgatg tgaagtacct catgaagagc tgcaaaaatc tcaaaggtgg
     4261 attacaggag gtggcagaac agttagagct ggaacggata ggaccacaac atcaggcagg
     4321 atctgattca ttgctcacag gaatggcctt tttcaaaatg agagaaatgt tctttgaaga
     4381 tcatattgat gatgccaaat attgtggtca tttgtatggc cttggttctg gttcatccta
     4441 tgtacagaat ggcacaggga atgcatatga agaggaagcc aacaagcagt catgaggatc
     4501 ctaatagtct agagctagcc ctaggagatc cggctgctaa caaagcccga aaggaagctg
     4561 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     4621 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
     4681 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     4741 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     4801 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tacaacttat
     4861 atcgtatggg gctgacttca ggtgctacat ttgaagagat aaattgcact gaaatcaaga
     4921 aatattttat ctgattaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct
     4981 cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta
     5041 ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt
     5101 cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg
     5161 gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg
     5221 gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga
     5281 actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg
     5341 aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg
     5401 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat
     5461 ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc
     5521 acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg ttcgtaagcc
     5581 atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa
     5641 tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct cctgccacat
     5701 gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata cactccccta
     5761 gcgctgatgt ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga
     5821 gggacagctg atagaaacag aagccactgg agcacctcaa aaacaccatc atacactaaa
     5881 tcagtaagtt ggcagcatca cccgacgcac tttgcgccga ataaatacct gtgacggaag
     5941 atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca
     6001 acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg
     6061 aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag
     6121 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta
     6181 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc
     6241 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct
     6301 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attc
//
