LOCUS       sequence-420963-        8293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4_64-575.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8293)
  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 8293)
  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_64-575_AG
FEATURES             Location/Qualifiers
     source          1..8293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        205..584
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        585..796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     747..767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     793..817
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          957..1089
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1072..1091
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1134..1152
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1165..1174
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1174..1215
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1237..1350
                     /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      2332..2341
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4064..4185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4075..4094)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4129..4148
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4371..4826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4458..4477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4668..4689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4943..5300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4943..5134
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4966..4986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5151..5286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5351..6145
                     /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(5405..5424)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6015..6034
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6209..6257
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6298..6317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6417..6439
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6477..6495)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6563..6667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6668..6736,6737..7528)
                     /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(6886..6905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7699..8287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8188..8207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catggctcga
       61 cagatcttca atattggcca ttagccatat tattcattgg ttatatagca taaatcaata
      121 ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct
      181 catgtccaat atgaccgcca tgttggcatt gattattgac tagttattaa tagtaatcaa
      241 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      301 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      361 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      421 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
      481 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
      541 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      601 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      661 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      721 acaactgcga tcgcccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
      781 ctatataagc agagctcgtt tagtgaaccg tcagatcact agaagcttta ttgcggtagt
      841 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
      901 ctgcagtgac tctcttaagg tagccttgca gaagttggtc gtgaggcact gggcaggtaa
      961 gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga
     1021 gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc
     1081 tctccacagg tgtccactcc cagttcaatt acagctctta aggctagagt acttaatacg
     1141 actcactata ggctagcaat tgcggccacc atgggtaagc ctatccctaa ccctctcctc
     1201 ggtctcgatt ctacgcccgg aagcggtagc ggtaccatgg acgagaagac caccggctgg
     1261 gggggcggcc acgtggtgga gggcctggcc ggcgagctgg agcagctgcg ggccaggctg
     1321 gagcaccacc ctcagggcca gcgggagccc tccggcggct gcaagcccct cgacatgaaa
     1381 atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct
     1441 gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat
     1501 aaactggaag agaaattccc gcaggttgcg gcaactggcg atggccctga cattatcttc
     1561 tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg
     1621 gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc
     1681 aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg
     1741 ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg
     1801 aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt
     1861 gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg
     1921 ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaagc
     1981 aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa
     2041 acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat
     2101 tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg
     2161 ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga gttcctcgaa
     2221 aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc
     2281 gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccaccatg
     2341 gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat
     2401 gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg
     2461 aaagaccctc cactcgagcc agcagtttat aaaccactct cccaggaaga gctgcaaagg
     2521 ataaagaatg agaaaaaaca gaaacaaaat gagagaaaac agaaaatatc agaaaatcgc
     2581 aaacatttgg ctagtgtacg tgtcgtacaa aaaaacctcg tctttgttgt aggtttatct
     2641 cagcgcctag cagacccaga ggttttaaaa cgaccagaat attttgggaa gtttggtaaa
     2701 atacataaag ttgtcatcaa taatagcaca tcatatgcag gctcacaggg tccaagtgcc
     2761 agtgcttatg taacctatat ccggtcagaa gacgctctca gagccataca gtgtgtcaac
     2821 aatgtggtag tagatggcag aacacttaag gcatctctag gtacaacaaa atactgcagt
     2881 tacttcttaa agaatatgca gtgtccaaaa cctgactgca tgtatcttca tgaattgggg
     2941 gatgaggcgg ccagcttcac aaaagaggaa atgcaggcgg gtaaacacca agaatatgaa
     3001 cagaagctac ttcaagaatt atataaatta aatcccaatt ttcttcagct atctacgggt
     3061 tcagttgata aaaataagaa caaagtgaca ccactgcaga ggtacgatac ccccattgac
     3121 aaaccttcag attctctcag tatagggaac ggtgataatt cccagcagat atctaacagt
     3181 gatacgcctt caccaccacc tggtttgtca aaatccaatc cagtcatccc catcagttca
     3241 tccaatcaca gtgcacggtc cccttttgaa ggggcagtaa cagagtcaca gtcgttattc
     3301 tcagacaatt ttcgccatcc caaccctatc ccaagtgggc ttcctccttt ccccagctcc
     3361 ccacagacat ccagtgactg gcctacagca ccagaaccac agagcctctt cacatcagaa
     3421 acaatcccag tatcatcctc tacagactgg caagcagctt ttggctttgg ttcttctaaa
     3481 caaccagagg atgacttggg ttttgatccc ttcgatgtca ctcgaaaagc cttagcagac
     3541 ctgattgaga aggaactgtc cgttcaagac caaccttccc tttcgcccac atctcttcag
     3601 aactcctctt cacacactac aaccgccaaa ggtccaggct ctggattcct gcatcctgct
     3661 gcagctacaa atgccaattc tctcaatagt accttttcag tcttgcccca gaggttccct
     3721 caatttcagc agcaccgagc ggtttataat tcattcagtt ttccaggcca ggcagcccgc
     3781 tatccttgga tggcctttcc acgcaatagc atcatgcact tgaaccacac agcaaacccc
     3841 acctcaaata gtaatttctt ggacttgaat ctcccgccac agcacaacac aggtctggga
     3901 gggatccctg tagcagggga agaagaggtg aaggtttcga ccatgccact gtcaacctct
     3961 tcccattcat tacaacaagg acagcagcct acaagtctcc acactactgt ggcctaagcg
     4021 gccgcttccc tttagtgagg gttaatgctt cgagcagaca tgataagata cattgatgag
     4081 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4141 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4201 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     4261 ctctacaaat gtggtaaaat ccgataagga tcgatccggg ctggcgtaat agcgaagagg
     4321 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     4381 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4441 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4501 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4561 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4621 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4681 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4741 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4801 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     4861 ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata acctctgaaa
     4921 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4981 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5041 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5101 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     5161 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5221 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5281 gaggcctagg cttttgcaaa aagcttgatt cttctgacac aacagtctcg aacttaaggc
     5341 tagagccacc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     5401 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     5461 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     5521 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     5581 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     5641 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     5701 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     5761 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     5821 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     5881 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5941 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     6001 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     6061 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     6121 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     6181 acgcccaacc tgccatcacg atggccgcaa taaaatatct ttattttcat tacatctgtg
     6241 tgttggtttt ttgtgtgaat cgatagcgat aaggatccgc gtatggtgca ctctcagtac
     6301 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     6361 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6421 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     6481 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     6541 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6601 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6661 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6721 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6781 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6841 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6901 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6961 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     7021 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     7081 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7141 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7201 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7261 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7321 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7381 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7441 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7501 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7561 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7621 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7681 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7741 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7801 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     7861 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7921 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7981 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     8041 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     8101 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8161 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8221 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8281 atggaaaaac gcc
//
