LOCUS       sequence-291228-        8116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4-del64-189.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8116)
  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 8116)
  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-del64-189-GSSG_AG
FEATURES             Location/Qualifiers
     source          1..8116
                     /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    3820..3941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3831..3850)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3885..3904
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4127..4582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4214..4233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4424..4445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4699..5056
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4699..4890
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4722..4742)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4907..5042
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5107..5901
                     /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(5161..5180)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5771..5790
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5965..6013
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6054..6073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6173..6195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6233..6251)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6319..6423
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6424..6492,6493..7284)
                     /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(6642..6661)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7455..8043
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7944..7963
                     /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 ggtagcagcg gtctaggtac aacaaaatac tgcagttact
     2641 tcttaaagaa tatgcagtgt ccaaaacctg actgcatgta tcttcatgaa ttgggggatg
     2701 aggcggccag cttcacaaaa gaggaaatgc aggcgggtaa acaccaagaa tatgaacaga
     2761 agctacttca agaattatat aaattaaatc ccaattttct tcagctatct acgggttcag
     2821 ttgataaaaa taagaacaaa gtgacaccac tgcagaggta cgataccccc attgacaaac
     2881 cttcagattc tctcagtata gggaacggtg ataattccca gcagatatct aacagtgata
     2941 cgccttcacc accacctggt ttgtcaaaat ccaatccagt catccccatc agttcatcca
     3001 atcacagtgc acggtcccct tttgaagggg cagtaacaga gtcacagtcg ttattctcag
     3061 acaattttcg ccatcccaac cctatcccaa gtgggcttcc tcctttcccc agctccccac
     3121 agacatccag tgactggcct acagcaccag aaccacagag cctcttcaca tcagaaacaa
     3181 tcccagtatc atcctctaca gactggcaag cagcttttgg ctttggttct tctaaacaac
     3241 cagaggatga cttgggtttt gatcccttcg atgtcactcg aaaagcctta gcagacctga
     3301 ttgagaagga actgtccgtt caagaccaac cttccctttc gcccacatct cttcagaact
     3361 cctcttcaca cactacaacc gccaaaggtc caggctctgg attcctgcat cctgctgcag
     3421 ctacaaatgc caattctctc aatagtacct tttcagtctt gccccagagg ttccctcaat
     3481 ttcagcagca ccgagcggtt tataattcat tcagttttcc aggccaggca gcccgctatc
     3541 cttggatggc ctttccacgc aatagcatca tgcacttgaa ccacacagca aaccccacct
     3601 caaatagtaa tttcttggac ttgaatctcc cgccacagca caacacaggt ctgggaggga
     3661 tccctgtagc aggggaagaa gaggtgaagg tttcgaccat gccactgtca acctcttccc
     3721 attcattaca acaaggacag cagcctacaa gtctccacac tactgtggcc taagcggccg
     3781 cttcccttta gtgagggtta atgcttcgag cagacatgat aagatacatt gatgagtttg
     3841 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3901 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3961 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     4021 acaaatgtgg taaaatccga taaggatcga tccgggctgg cgtaatagcg aagaggcccg
     4081 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc
     4141 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4201 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4261 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4321 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4381 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4441 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4501 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4561 aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4621 tttcacaccg catacgcgga tctgcgcagc accatggcct gaaataacct ctgaaagagg
     4681 aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     4741 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4801 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4861 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4921 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4981 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     5041 cctaggcttt tgcaaaaagc ttgattcttc tgacacaaca gtctcgaact taaggctaga
     5101 gccaccatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     5161 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     5221 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     5281 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     5341 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5401 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5461 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     5521 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5581 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     5641 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5701 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5761 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5821 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5881 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5941 ccaacctgcc atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt
     6001 ggttttttgt gtgaatcgat agcgataagg atccgcgtat ggtgcactct cagtacaatc
     6061 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     6121 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     6181 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     6241 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     6301 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6361 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6421 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6481 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6541 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6601 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6661 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6721 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6781 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6841 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6901 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6961 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     7021 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     7081 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     7141 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     7201 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     7261 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     7321 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     7381 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7441 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7501 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7561 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     7621 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7681 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7741 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7801 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7861 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7921 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7981 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     8041 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     8101 atggctcgac agatct
//
