LOCUS       sequence-259917-        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"
     primer_bind     complement(59..81)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(226..244)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        312..416
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(417..485,486..1277)
                     /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(635..654)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1448..2036
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1937..1956
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2190..2207
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2280..2357
                     /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             2358..3440
                     /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(2377..2396)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    3453..3474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3489..3494,3495..3512,3513..3519)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3527..3543
                     /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     3551..3567
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3551..3567
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3563..4090
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(3583..3600)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3583..3599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3592..3614)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(4192..4197,4198..4213,4214..4220)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4228..4244
                     /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             4267..4920
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     4878..4900
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             4927..4950
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4960..5073
                     /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"
ORIGIN
        1 ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
       61 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
      121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
      181 agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc gtgatacgcc
      241 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
      301 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
      361 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
      421 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
      481 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
      541 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
      601 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
      661 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
      721 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
      781 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
      841 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
      901 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
      961 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1021 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1081 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     1141 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1201 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1261 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     1321 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     1381 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1441 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1501 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1561 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1621 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1681 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1741 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1801 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1861 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1921 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1981 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2041 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2101 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2161 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2221 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataaattccg
     2281 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     2341 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     2401 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2461 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2521 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2581 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2641 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2701 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     2761 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     2821 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     2881 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     2941 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     3001 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     3061 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     3121 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     3181 ataccgaaga cagctcatgt tatatcccgc cgtcaaccac catcaaacag gattttcgcc
     3241 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     3301 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     3361 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3421 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3481 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3541 caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca
     3601 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     3661 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     3721 cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag
     3781 ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat
     3841 gcacggttac gatgcgccca tctacaccaa cgtaacctat cccattacgg tcaatccgcc
     3901 gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag
     3961 ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta cgttatcgac
     4021 tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg
     4081 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg
     4141 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt
     4201 aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca
     4261 gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga
     4321 cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt
     4381 gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat
     4441 attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag
     4501 cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg
     4561 gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc
     4621 aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt
     4681 cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct
     4741 cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt
     4801 tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat
     4861 atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa
     4921 tcggatctgg aagttctgtt ccaggggccc ctgggattaa tggacgaaaa aaccaccggt
     4981 tggcgtggtg gtcacgttgt tgaaggtctg gctggtgaac tggaacagct gcgtgctcgt
     5041 ctggaacacc acccgcaggg tcagcgtgaa ccgggatccc catatccaga agacccagca
     5101 gtttataaac cactctccca ggaagagctg caaaggataa agaatgagaa aaaacagaaa
     5161 caaaatgaga gaaaacagaa aatatcagaa aatcgcaaac atttggctag tgtacgtgtc
     5221 gtacaaaaaa acctcgtctt tgttgtaggt ttatctcagc gcctagcaga cccagaggtt
     5281 ttaaaacgac cagaatattt tgggaagttt ggtaaaatac ataaagttgt catcaataat
     5341 agcacatcat atgcaggctc acagggtcca agtgccagtg cttatgtaac ctatatccgg
     5401 tcagaagacg ctctcagagc catacagtgt gtcaacaatg tggtagtaga tggcagaaca
     5461 cttaaggcat ctctaggtac aacaaaatac tgcagttact tcttaaagaa tatgcagtgt
     5521 ccaaaacctg actgcatgta tcttcatgaa ttgggggatg aggcggccag cttcacaaaa
     5581 gaggaaatgc aggcgggtaa acaccaagaa tatgaacaga agctacttca agaattatat
     5641 aaattaaatc ccaattttct tcagctatct acgggttcag ttgattgagc
//
