LOCUS       sequence-290169-        5233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot3_666-753.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5233)
  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 5233)
  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: pGEX-6P-1-HsNot3_666-753_U
FEATURES             Location/Qualifiers
     source          1..5233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1283..1305)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1450..1468)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1536..1640
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1641..1709,1710..2501)
                     /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(1859..1878)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2672..3260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3161..3180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3414..3431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3504..3581
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3582..4664
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3601..3620)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4677..4698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4713..4718,4719..4736,4737..4743)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4751..4767
                     /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)."
     primer_bind     4775..4791
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4775..4791
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4787..5233,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4807..4824)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4807..4823)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4816..4838)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ctgtcgaccg
      961 agacactctt cttcatcttc tactatctgg agggcactaa ggcacagtat ctggcagcca
     1021 aggccctaaa gaagcagtca tggcgattcc acaccaagta catgatgtgg ttccagaggc
     1081 acgaggagcc caagaccatc actgacgagt ttgagcaggg cacctacatc tactttgact
     1141 acgagaagtg gggccagcgg aagaaggaag gcttcacctt tgagtaccgc tacctggagg
     1201 accgggacct ccagtaactc gagcggccgc atcgtgactg actgacgatc tgcctcgcgc
     1261 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     1321 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     1381 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa
     1441 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1501 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1561 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1621 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1681 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1741 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1801 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1861 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1921 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1981 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2041 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2101 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2161 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2221 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2281 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2341 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2401 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2461 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2521 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2581 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2641 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2701 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2761 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2821 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2881 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2941 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3001 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3061 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3301 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3361 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3421 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3481 gtatttcaca ccgcataaat tccgacacca tcgaatggtg caaaaccttt cgcggtatgg
     3541 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     3601 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     3661 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     3721 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     3781 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     3841 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     3901 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     3961 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     4021 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     4081 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     4141 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     4201 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     4261 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     4321 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     4381 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgtcaa
     4441 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     4501 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     4561 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4621 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4681 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     4741 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     4801 ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4861 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4921 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc
     4981 ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt
     5041 cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtaac
     5101 ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc
     5161 gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga
     5221 tggcgttgga att
//
