LOCUS       sequence-291213-        7795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4_64-104-GSSG_275-575.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7795)
  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 7795)
  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_64-104-GSSG-275-575_AG
FEATURES             Location/Qualifiers
     source          1..7795
                     /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    3499..3620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3510..3529)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3564..3583
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3806..4261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3893..3912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4103..4124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4378..4735
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4378..4569
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4401..4421)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4586..4721
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4786..5580
                     /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(4840..4859)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5450..5469
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5644..5692
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5733..5752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5852..5874
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5912..5930)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5998..6102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6103..6171,6172..6963)
                     /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(6321..6340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7134..7722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7623..7642
                     /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 gccagcagtt tataaaccac tctcccagga agagctgcaa aggataaaga
     2461 atgagaaaaa acagaaacaa aatgagagaa aacagaaaat atcagaaaat cgcaaacatt
     2521 tggctagtgt acgtggtagc agcggtccca ttgacaaacc ttcagattct ctcagtatag
     2581 ggaacggtga taattcccag cagatatcta acagtgatac gccttcacca ccacctggtt
     2641 tgtcaaaatc caatccagtc atccccatca gttcatccaa tcacagtgca cggtcccctt
     2701 ttgaaggggc agtaacagag tcacagtcgt tattctcaga caattttcgc catcccaacc
     2761 ctatcccaag tgggcttcct cctttcccca gctccccaca gacatccagt gactggccta
     2821 cagcaccaga accacagagc ctcttcacat cagaaacaat cccagtatca tcctctacag
     2881 actggcaagc agcttttggc tttggttctt ctaaacaacc agaggatgac ttgggttttg
     2941 atcccttcga tgtcactcga aaagccttag cagacctgat tgagaaggaa ctgtccgttc
     3001 aagaccaacc ttccctttcg cccacatctc ttcagaactc ctcttcacac actacaaccg
     3061 ccaaaggtcc aggctctgga ttcctgcatc ctgctgcagc tacaaatgcc aattctctca
     3121 atagtacctt ttcagtcttg ccccagaggt tccctcaatt tcagcagcac cgagcggttt
     3181 ataattcatt cagttttcca ggccaggcag cccgctatcc ttggatggcc tttccacgca
     3241 atagcatcat gcacttgaac cacacagcaa accccacctc aaatagtaat ttcttggact
     3301 tgaatctccc gccacagcac aacacaggtc tgggagggat ccctgtagca ggggaagaag
     3361 aggtgaaggt ttcgaccatg ccactgtcaa cctcttccca ttcattacaa caaggacagc
     3421 agcctacaag tctccacact actgtggcct aagcggccgc ttccctttag tgagggttaa
     3481 tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3541 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3601 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3661 gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatccgat
     3721 aaggatcgat ccgggctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3781 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3841 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3901 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3961 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4021 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4081 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4141 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4201 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4261 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgcggat
     4321 ctgcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg
     4381 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4441 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4501 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4561 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4621 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4681 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4741 tgattcttct gacacaacag tctcgaactt aaggctagag ccaccatgat tgaacaagat
     4801 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4861 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4921 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4981 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     5041 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     5101 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5161 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5221 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5281 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     5341 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5401 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5461 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5521 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5581 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc
     5641 cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata
     5701 gcgataagga tccgcgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5761 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5821 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5881 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5941 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6001 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6061 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6121 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6181 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6241 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6301 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6361 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6421 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6481 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6541 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6601 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6661 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6721 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6781 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6841 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6901 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6961 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7021 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7081 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7141 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7201 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7261 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7321 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7381 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7441 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7501 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7561 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7621 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7681 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7741 tttttacggt tcctggcctt ttgctggcct tttgctcaca tggctcgaca gatct
//
