LOCUS       sequence-291229-        8251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4-del109-189.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8251)
  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 8251)
  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-del109-189-GSSG_AG
FEATURES             Location/Qualifiers
     source          1..8251
                     /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    3955..4076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3966..3985)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4020..4039
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4262..4717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4349..4368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4559..4580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4834..5191
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4834..5025
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4857..4877)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5042..5177
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5242..6036
                     /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(5296..5315)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5906..5925
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6100..6148
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6189..6208)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6308..6330
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6368..6386)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6454..6558
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6559..6627,6628..7419)
                     /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(6777..6796)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7590..8178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8079..8098
                     /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 gatgtctcgc agtcctgatg cgaaggaaga ccctgtggag tgccctcttt
     2461 gcatggagcc cttggagata gatgatatca actttttccc ttgcacctgt ggctaccaga
     2521 tttgccgatt ttgttggcat cgaattcgca ctgatgaaaa tgggctttgt cctgcatgta
     2581 gaaagccata tccagaagac ccagcagttt ataaaccact ctcccaggaa gagctgcaaa
     2641 ggataaagaa tgagaaaaaa cagaaacaaa atgagagaaa acagaaaata tcagaaaatc
     2701 gcaaacattt ggctagtgta cgtgtcgtac aaaaaggtag cagcggtcta ggtacaacaa
     2761 aatactgcag ttacttctta aagaatatgc agtgtccaaa acctgactgc atgtatcttc
     2821 atgaattggg ggatgaggcg gccagcttca caaaagagga aatgcaggcg ggtaaacacc
     2881 aagaatatga acagaagcta cttcaagaat tatataaatt aaatcccaat tttcttcagc
     2941 tatctacggg ttcagttgat aaaaataaga acaaagtgac accactgcag aggtacgata
     3001 cccccattga caaaccttca gattctctca gtatagggaa cggtgataat tcccagcaga
     3061 tatctaacag tgatacgcct tcaccaccac ctggtttgtc aaaatccaat ccagtcatcc
     3121 ccatcagttc atccaatcac agtgcacggt ccccttttga aggggcagta acagagtcac
     3181 agtcgttatt ctcagacaat tttcgccatc ccaaccctat cccaagtggg cttcctcctt
     3241 tccccagctc cccacagaca tccagtgact ggcctacagc accagaacca cagagcctct
     3301 tcacatcaga aacaatccca gtatcatcct ctacagactg gcaagcagct tttggctttg
     3361 gttcttctaa acaaccagag gatgacttgg gttttgatcc cttcgatgtc actcgaaaag
     3421 ccttagcaga cctgattgag aaggaactgt ccgttcaaga ccaaccttcc ctttcgccca
     3481 catctcttca gaactcctct tcacacacta caaccgccaa aggtccaggc tctggattcc
     3541 tgcatcctgc tgcagctaca aatgccaatt ctctcaatag taccttttca gtcttgcccc
     3601 agaggttccc tcaatttcag cagcaccgag cggtttataa ttcattcagt tttccaggcc
     3661 aggcagcccg ctatccttgg atggcctttc cacgcaatag catcatgcac ttgaaccaca
     3721 cagcaaaccc cacctcaaat agtaatttct tggacttgaa tctcccgcca cagcacaaca
     3781 caggtctggg agggatccct gtagcagggg aagaagaggt gaaggtttcg accatgccac
     3841 tgtcaacctc ttcccattca ttacaacaag gacagcagcc tacaagtctc cacactactg
     3901 tggcctaagc ggccgcttcc ctttagtgag ggttaatgct tcgagcagac atgataagat
     3961 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     4021 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     4081 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     4141 gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg gctggcgtaa
     4201 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4261 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4321 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4381 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4441 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4501 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4561 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4621 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4681 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     4741 cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat
     4801 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     4861 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4921 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4981 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5041 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5101 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5161 aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca caacagtctc
     5221 gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg ttctccggcc
     5281 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5341 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5401 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5461 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5521 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5581 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5641 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5701 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5761 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5821 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5881 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5941 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     6001 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     6061 ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc tttattttca
     6121 ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg cgtatggtgc
     6181 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6241 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6301 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     6361 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6421 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6481 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6541 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6601 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6661 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6721 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6781 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6841 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6901 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6961 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     7021 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     7081 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     7141 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7201 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7261 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7321 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7381 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7441 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7501 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7561 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7621 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7681 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7741 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7801 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7861 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7921 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7981 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     8041 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     8101 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     8161 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     8221 tggccttttg ctcacatggc tcgacagatc t
//
