LOCUS       sequence-275067-        5690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of CNOT4 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5690)
  AUTHORS   Dominguez D, Freese P, Alexis MS, Su A, Hochman M, Palden T, Bazile 
            C, Lambert NJ, Van Nostrand EL, Pratt GA, Yeo GW, Graveley BR, Burge
            CB
  TITLE     Sequence, Structure, and Context Preferences of Human RNA Binding 
            Proteins.
  JOURNAL   Mol Cell. 2018 Jun 7;70(5):854-867.e9. doi: 
            10.1016/j.molcel.2018.05.001. Epub 2018 Jun 7.
  PUBMED    29883606
REFERENCE   2  (bases 1 to 5690)
  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.2018.05.001"; journalName: "Mol Cell"; date: 
            "2018-06-7- 7"; volume: "70"; issue: "5"; pages: "854-867.e9"
COMMENT     Sequence Label: CNOT4_RRM-ZNF_pGEX
FEATURES             Location/Qualifiers
     source          1..5690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             769..882
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     primer_bind     complement(1558..1580)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1725..1743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1811..1915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1916..1984,1985..2776)
                     /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(2134..2153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2947..3535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3436..3455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3689..3706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3779..3856
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3857..4939
                     /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     complement(3876..3895)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4988..4993,4994..5011,5012..5018)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5026..5042
                     /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     5050..5066
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5050..5066
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5062..5589
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5082..5099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5082..5098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5091..5113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggattaat ggacgaaaaa
      781 accaccggtt ggcgtggtgg tcacgttgtt gaaggtctgg ctggtgaact ggaacagctg
      841 cgtgctcgtc tggaacacca cccgcagggt cagcgtgaac cgggatcccc atatccagaa
      901 gacccagcag tttataaacc actctcccag gaagagctgc aaaggataaa gaatgagaaa
      961 aaacagaaac aaaatgagag aaaacagaaa atatcagaaa atcgcaaaca tttggctagt
     1021 gtacgtgtcg tacaaaaaaa cctcgtcttt gttgtaggtt tatctcagcg cctagcagac
     1081 ccagaggttt taaaacgacc agaatatttt gggaagtttg gtaaaataca taaagttgtc
     1141 atcaataata gcacatcata tgcaggctca cagggtccaa gtgccagtgc ttatgtaacc
     1201 tatatccggt cagaagacgc tctcagagcc atacagtgtg tcaacaatgt ggtagtagat
     1261 ggcagaacac ttaaggcatc tctaggtaca acaaaatact gcagttactt cttaaagaat
     1321 atgcagtgtc caaaacctga ctgcatgtat cttcatgaat tgggggatga ggcggccagc
     1381 ttcacaaaag aggaaatgca ggcgggtaaa caccaagaat atgaacagaa gctacttcaa
     1441 gaattatata aattaaatcc caattttctt cagctatcta cgggttcagt tgattgagcg
     1501 gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     1561 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     1621 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     1681 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     1741 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1801 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1861 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1921 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1981 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2041 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2101 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2161 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2221 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2281 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2341 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2401 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2461 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2521 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2581 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2641 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2701 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2761 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2821 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2881 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2941 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3001 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3061 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3121 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3181 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3241 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3301 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3361 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3421 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3481 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3541 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3601 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3661 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3721 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga
     3781 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     3841 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     3901 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     3961 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4021 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4081 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4141 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     4201 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     4261 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     4321 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     4381 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     4441 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     4501 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     4561 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     4621 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     4681 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     4741 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     4801 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc acgctagcgc ccaatacgca
     4861 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4921 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     4981 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5041 aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa
     5101 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5161 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5221 agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc
     5281 tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg
     5341 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg
     5401 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
     5461 tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaattag cttatcgact
     5521 gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt atggctgtgc
     5581 aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt
     5641 tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
//
