LOCUS       sequence-291224-        7660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4_275-575-L415E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7660)
  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 7660)
  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_275-575-L415E_AG
FEATURES             Location/Qualifiers
     source          1..7660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1098..1107
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1107..1148
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1170..1283
                     /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"
     regulatory      2265..2274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3364..3485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3375..3394)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3429..3448
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3671..4126
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3758..3777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3968..3989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4243..4600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4243..4434
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4266..4286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4451..4586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4651..5445
                     /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(4705..4724)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5315..5334
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5509..5557
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5598..5617)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5717..5739
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5777..5795)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5863..5967
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5968..6036,6037..6828)
                     /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"
     primer_bind     complement(6186..6205)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6999..7587
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7488..7507
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc aattgcggcc accatgggta agcctatccc taaccctctc ctcggtctcg
     1141 attctacgcc cggaagcggt agcggtacca tggacgagaa gaccaccggc tgggggggcg
     1201 gccacgtggt ggagggcctg gccggcgagc tggagcagct gcgggccagg ctggagcacc
     1261 accctcaggg ccagcgggag ccctccggcg gctgcaagcc cctcgacatg aaaatcgaag
     1321 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     1381 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     1441 aagagaaatt cccgcaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     1501 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     1561 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     1621 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     1681 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     1741 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
     1801 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
     1861 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa agcaaacaca
     1921 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
     1981 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
     2041 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
     2101 caggtattaa cgccgccagt ccgaacaaag agctggcgaa agagttcctc gaaaactatc
     2161 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     2221 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc atggaaaacg
     2281 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     2341 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacc
     2401 ctccactcga gcccattgac aaaccttcag attctctcag tatagggaac ggtgataatt
     2461 cccagcagat atctaacagt gatacgcctt caccaccacc tggtttgtca aaatccaatc
     2521 cagtcatccc catcagttca tccaatcaca gtgcacggtc cccttttgaa ggggcagtaa
     2581 cagagtcaca gtcgttattc tcagacaatt ttcgccatcc caaccctatc ccaagtgggc
     2641 ttcctccttt ccccagctcc ccacagacat ccagtgactg gcctacagca ccagaaccac
     2701 agagcctctt cacatcagaa acaatcccag tatcatcctc tacagactgg caagcagctt
     2761 ttggctttgg ttcttctaaa caaccagagg atgacttggg ttttgatccc ttcgatgtca
     2821 ctcgaaaagc cgaggcagac ctgattgaga aggaactgtc cgttcaagac caaccttccc
     2881 tttcgcccac atctcttcag aactcctctt cacacactac aaccgccaaa ggtccaggct
     2941 ctggattcct gcatcctgct gcagctacaa atgccaattc tctcaatagt accttttcag
     3001 tcttgcccca gaggttccct caatttcagc agcaccgagc ggtttataat tcattcagtt
     3061 ttccaggcca ggcagcccgc tatccttgga tggcctttcc acgcaatagc atcatgcact
     3121 tgaaccacac agcaaacccc acctcaaata gtaatttctt ggacttgaat ctcccgccac
     3181 agcacaacac aggtctggga gggatccctg tagcagggga agaagaggtg aaggtttcga
     3241 ccatgccact gtcaacctct tcccattcat tacaacaagg acagcagcct acaagtctcc
     3301 acactactgt ggcctaagcg gccgcttccc tttagtgagg gttaatgctt cgagcagaca
     3361 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3421 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3481 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     3541 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat ccgataagga tcgatccggg
     3601 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     3661 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3721 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3781 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3841 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3901 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3961 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4021 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4081 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt
     4141 ttctccttac gcatctgtgc ggtatttcac accgcatacg cggatctgcg cagcaccatg
     4201 gcctgaaata acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca
     4261 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4321 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4441 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4501 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4561 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttgatt cttctgacac
     4621 aacagtctcg aacttaaggc tagagccacc atgattgaac aagatggatt gcacgcaggt
     4681 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4741 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4801 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4861 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4921 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4981 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5041 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5101 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5161 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5221 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5281 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5341 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5401 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5461 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg atggccgcaa taaaatatct
     5521 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagcgat aaggatccgc
     5581 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5641 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5701 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5761 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5821 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5881 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5941 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6001 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6061 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6121 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6181 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6241 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6301 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6361 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6421 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6481 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6541 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6601 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6661 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6721 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6781 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6841 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6901 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6961 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7021 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7081 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7141 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7201 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7261 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7321 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7381 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7441 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7501 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7561 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7621 gccttttgct ggccttttgc tcacatggct cgacagatct
//
