LOCUS       sequence-290782-        7967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot3_607-748-Y702A-HsNot2_350-540.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7967)
  AUTHORS   Raisch T, Bhandari D, Sabath K, Helms S, Valkov E, Weichenrieder O, 
            Izaurralde E
  TITLE     Distinct modes of recruitment of the CCR4-NOT complex by Drosophila 
            and vertebrate Nanos.
  JOURNAL   EMBO J. 2016 May 2;35(9):974-90. doi: 10.15252/embj.201593634. Epub 
            2016 Mar 11.
  PUBMED    26968986
REFERENCE   2  (bases 1 to 7967)
  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: 
            "10.15252/embj.201593634"; journalName: "EMBO J"; date: "2016-05-2- 
            2"; volume: "35"; issue: "9"; pages: "974-90"
COMMENT     Sequence Label: pnEA-NpH-HsNot3_607-748-Y702A-NpM-HsNot2_350-5...
FEATURES             Location/Qualifiers
     source          1..7967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2082..2101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2242..2264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3761..4843)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4805..4824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4844..4921)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5127..5146
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5230..5249
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5230..5248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5249..5273
                     /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             5288..5310
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5329..5346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5368..5391
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             5857..5879
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5886..6986
                     /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             7002..7025
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(7694..7712)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7708..7755
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7930..7935,7936..7952,7953..7958))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     4141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5041 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5101 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     5161 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     5221 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tccccactag
     5281 taataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     5341 catcacagca gcggtaccgg cagcggcctg gaagttctgt tccaggggcc ccatatgctc
     5401 gagctcacca aggagcagct ctatcagcag gccatggaag aggccgcctg gcaccacatg
     5461 cctcacccct ctgactctga gcgtattcgg cagtacctcc cccggaaccc ctgtccgacg
     5521 cccccctacc accaccagat gccaccccca cactcggaca ctgtggaatt ctaccagcgc
     5581 ctgtcgaccg agacactctt cttcatcttc tactatctgg agggcactaa ggcacagtat
     5641 ctggcagcca aggccctaaa gaagcagtca tggcgattcc acaccaaggc gatgatgtgg
     5701 ttccagaggc acgaggagcc caagaccatc actgacgagt ttgagcaggg cacctacatc
     5761 tactttgact acgagaagtg gggccagcgg aagaaggaag gcttcacctt tgagtaccgc
     5821 tacctggagt aaggatccta atagtctagt aataattttg tttaacttta agaaggagat
     5881 ataccatgga aactgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata
     5941 acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg
     6001 agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg
     6061 acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg
     6121 aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac
     6181 gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata
     6241 acaaagacct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag
     6301 aactgaaagc gaaaggtaag agcgccctga tgttcaacct gcaagaaccg tacttcacct
     6361 ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca
     6421 ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc
     6481 tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta
     6541 ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca
     6601 gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt
     6661 tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag
     6721 agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac
     6781 cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg
     6841 ccgccacgat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg
     6901 ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg
     6961 atgaagccct taaagacgcg cagactggta ccggcagcgg cctggaagtt ctgttccagg
     7021 ggccccatat gctcgagatg gtgacggacc aatttggaat gattggcctg ttaacattta
     7081 tcagggcagc agagacagac ccaggaatgg tacatcttgc attaggaagt gacttaacaa
     7141 cattaggcct caatctgaac tctcctgaaa atctctaccc caaatttgcg tcaccctggg
     7201 catcttcacc ttgtcgacct caagacatag acttccatgt tccatctgag tacttaacga
     7261 acattcacat tagggataag ctggctgcaa taaaacttgg ccgatatggt gaagaccttc
     7321 tcttctatct ctattacatg aatggaggag acgtattaca acttttagct gcagtggagc
     7381 tttttaaccg tgattggaga taccacaaag aagaacgagt atggattacc agggcaccag
     7441 gcatggagcc aacaatgaaa accaatacct atgagagggg aacatattac ttctttgact
     7501 gtcttaactg gaggaaagta gctaaggagt tccatctgga atatgacaaa ttagaagaac
     7561 ggcctcacct gccatccacc ttcaactaca accctgctca gcaagccttc taaggatcct
     7621 aatagtctag agctagccct aggagatccg gctgctaaca aagcccgaaa ggaagctgag
     7681 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     7741 ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca
     7801 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     7861 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     7921 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaa
//
