LOCUS       sequence-420959-        8482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8482)
  AUTHORS   Keskeny C, Raisch T, Sgromo A, Igreja C, Bhandari D, Weichenrieder 
            O, Izaurralde E
  TITLE     A conserved CAF40-binding motif in metazoan NOT4 mediates 
            association with the CCR4-NOT complex.
  JOURNAL   Genes Dev. 2019 Feb 1;33(3-4):236-252. doi: 10.1101/gad.320952.118. 
            Epub 2019 Jan 28.
  PUBMED    30692204
REFERENCE   2  (bases 1 to 8482)
  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"; journalName: "Genes 
            Dev."; date: "2019-02-1"; volume: "33(3-4):236-252. doi"; pages: " 
            10.1101/gad.320952.118. Epub 2019 Jan 28"
COMMENT     Sequence Label: pCIneo-V5-SBP-MBP-HsNot4_AG
FEATURES             Location/Qualifiers
     source          1..8482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     191..210
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(429..533)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     601..619
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(657..679)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     779..798
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    839..887
                     /note="synthetic polyadenylation signal"
     CDS             complement(951..1745)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1062..1081)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1672..1691
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1796..2153)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1810..1945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        1962..2153
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2110..2130
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2270..2725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2407..2428)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2619..2638
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2911..3032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2948..2967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3002..3021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     regulatory      4944..4953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5935..6048)
                     /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="MDEKTTGWGGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             complement(6070..6111)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     regulatory      6111..6120
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6133..6151)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6194..6213)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          6196..6328
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(6468..6492)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(6489..6700)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6518..6538)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6701..7080
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(7291..7879)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7371..7390)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8050..8482,1..359,360..428))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
       61 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      121 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      181 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      241 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      301 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      361 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      421 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      481 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      541 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
      601 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
      661 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
      721 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
      781 gcatcagagc agattgtact gagagtgcac catacgcgga tccttatcgc tatcgattca
      841 cacaaaaaac caacacacag atgtaatgaa aataaagata ttttattgcg gccatcgtga
      901 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac
      961 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     1021 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     1081 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     1141 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     1201 tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     1261 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     1321 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     1381 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     1441 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     1501 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     1561 tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     1621 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     1681 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     1741 atcatggtgg ctctagcctt aagttcgaga ctgttgtgtc agaagaatca agctttttgc
     1801 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
     1861 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     1921 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     1981 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     2041 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     2101 actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac
     2161 ctaaccaagt tcctctttca gaggttattt caggccatgg tgctgcgcag atccgcgtat
     2221 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     2281 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2341 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2401 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2461 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     2521 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     2581 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     2641 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     2701 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     2761 ggcgatcggt gcgggcctct tcgctattac gccagcccgg atcgatcctt atcggatttt
     2821 accacatttg tagaggtttt acttgcttta aaaaacctcc cacatctccc cctgaacctg
     2881 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     2941 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3001 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gctcgaagca ttaaccctca
     3061 ctaaagggaa gcggccgctt aggccacagt agtgtggaga cttgtaggct gctgtccttg
     3121 ttgtaatgaa tgggaagagg ttgacagtgg catggtcgaa accttcacct cttcttcccc
     3181 tgctacaggg atccctccca gacctgtgtt gtgctgtggc gggagattca agtccaagaa
     3241 attactattt gaggtggggt ttgctgtgtg gttcaagtgc atgatgctat tgcgtggaaa
     3301 ggccatccaa ggatagcggg ctgcctggcc tggaaaactg aatgaattat aaaccgctcg
     3361 gtgctgctga aattgaggga acctctgggg caagactgaa aaggtactat tgagagaatt
     3421 ggcatttgta gctgcagcag gatgcaggaa tccagagcct ggacctttgg cggttgtagt
     3481 gtgtgaagag gagttctgaa gagatgtggg cgaaagggaa ggttggtctt gaacggacag
     3541 ttccttctca atcaggtctg ctaaggcttt tcgagtgaca tcgaagggat caaaacccaa
     3601 gtcatcctct ggttgtttag aagaaccaaa gccaaaagct gcttgccagt ctgtagagga
     3661 tgatactggg attgtttctg atgtgaagag gctctgtggt tctggtgctg taggccagtc
     3721 actggatgtc tgtggggagc tggggaaagg aggaagccca cttgggatag ggttgggatg
     3781 gcgaaaattg tctgagaata acgactgtga ctctgttact gccccttcaa aaggggaccg
     3841 tgcactgtga ttggatgaac tgatggggat gactggattg gattttgaca aaccaggtgg
     3901 tggtgaaggc gtatcactgt tagatatctg ctgggaatta tcaccgttcc ctatactgag
     3961 agaatctgaa ggtttgtcaa tgggggtatc gtacctctgc agtggtgtca ctttgttctt
     4021 atttttatca actgaacccg tagatagctg aagaaaattg ggatttaatt tatataattc
     4081 ttgaagtagc ttctgttcat attcttggtg tttacccgcc tgcatttcct cttttgtgaa
     4141 gctggccgcc tcatccccca attcatgaag atacatgcag tcaggttttg gacactgcat
     4201 attctttaag aagtaactgc agtattttgt tgtacctaga gatgccttaa gtgttctgcc
     4261 atctactacc acattgttga cacactgtat ggctctgaga gcgtcttctg accggatata
     4321 ggttacataa gcactggcac ttggaccctg tgagcctgca tatgatgtgc tattattgat
     4381 gacaacttta tgtattttac caaacttccc aaaatattct ggtcgtttta aaacctctgg
     4441 gtctgctagg cgctgagata aacctacaac aaagacgagg tttttttgta cgacacgtac
     4501 actagccaaa tgtttgcgat tttctgatat tttctgtttt ctctcatttt gtttctgttt
     4561 tttctcattc tttatccttt gcagctcttc ctgggagagt ggtttataaa ctgctgggtc
     4621 ttctggatat ggctttctac atgcaggaca aagcccattt tcatcagtgc gaattcgatg
     4681 ccaacaaaat cggcaaatct ggtagccaca ggtgcaaggg aaaaagttga tatcatctat
     4741 ctccaagggc tccatgcaaa gagggcactc cacagggtct tccttcgcat caggactgcg
     4801 agacatctcg agtggagggt ctttcagggc ttcatcgaca gtctgacgac cgctggcggc
     4861 gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga tgttcggcat
     4921 gatttcacct ttctgggcgt tttccatggt ggcggcaata cgtggatctt tcgccaactc
     4981 ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa ccgcttccag
     5041 accttcatca gtcagcagat agttttcgag gaactctttc gccagctctt tgttcggact
     5101 ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt gacccttgaa
     5161 ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg accatgccca
     5221 cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg cgatggagta
     5281 atcggtgtct gcattcatgt gtttgctttt aatcaggtca accaggaagg tcagacccgc
     5341 tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg tacttgccgt tttcatactt
     5401 gaacgcataa cccccgtcag cagcaatcag cggccaggtg aagtacggtt cttgcaggtt
     5461 gaacatcagc gcgctcttac ctttcgcttt cagttcttta tccagcgccg ggatctcttc
     5521 ccaggttttt ggcgggttcg gcagcagatc tttgttataa atcagcgata acgcttcaac
     5581 agcgatcggg taagcaatca gcttgccgtt gtaacgtacg gcatcccagg taaacggata
     5641 cagcttgtcc tggaacgctt tgtccggggt gatttcagcc aacaggccag attgagcgta
     5701 gccaccaaag cggtcgtgtg cccagaagat aatgtcaggg ccatcgccag ttgccgcaac
     5761 ctgcgggaat ttctcttcca gtttatccgg atgctcaacg gtgactttaa ttccggtatc
     5821 tttctcgaat ttcttaccga cttcagcgag accgttatag cctttatcgc cgttaatcca
     5881 gattaccagt ttaccttctt cgattttcat gtcgaggggc ttgcagccgc cggagggctc
     5941 ccgctggccc tgagggtggt gctccagcct ggcccgcagc tgctccagct cgccggccag
     6001 gccctccacc acgtggccgc ccccccagcc ggtggtcttc tcgtccatgg taccgctacc
     6061 gcttccgggc gtagaatcga gaccgaggag agggttaggg ataggcttac ccatggtggc
     6121 cgcaattgct agcctatagt gagtcgtatt aagtactcta gccttaagag ctgtaattga
     6181 actgggagtg gacacctgtg gagagaaagg caaagtggat gtcagtaaga ccaataggtg
     6241 cctatcagaa acgcaagagt cttctctgtc tcgacaagcc cagtttctat tggtctcctt
     6301 aaacctgtct tgtaaccttg atacttacct gcccagtgcc tcacgaccaa cttctgcaag
     6361 gctaccttaa gagagtcact gcagcttaag ttcgagactg ttgtgtcaga agcactgact
     6421 gcgttagcaa tttaactgtg ataaactacc gcaataaagc ttctagtgat ctgacggttc
     6481 actaaacgag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     6541 aacggggcgg gcgatcgcag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     6601 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     6661 gctatccacg cccattggtg tactgccaaa accgcatcac catggtaata gcgatgacta
     6721 atacgtagat gtactgccaa gtaggaaagt cccgtaaggt catgtactgg gcataatgcc
     6781 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgg acttggcata tgatacactt
     6841 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     6901 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     6961 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     7021 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgcc
     7081 aacatggcgg tcatattgga catgagccaa tataaatgta catattatga tatagataca
     7141 acgtatgcaa tggccaatag ccaatattga tttatgctat ataaccaatg aataatatgg
     7201 ctaatggcca atattgaaga tctgtcgagc catgtgagca aaaggccagc aaaaggccag
     7261 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     7321 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     7381 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     7441 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     7501 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     7561 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     7621 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     7681 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     7741 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7801 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7861 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7921 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7981 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     8041 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     8101 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     8161 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     8221 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     8281 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     8341 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     8401 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8461 caaaaaagcg gttagctcct tc
//
