LOCUS       sequence-316927-        6774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot4_275-575.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6774)
  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 6774)
  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: pcDNA3.1-MS2-HA-HsNot4_275-575_AG
FEATURES             Location/Qualifiers
     source          1..6774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1099..1125
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2093..2111)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2094..2111)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2131..2148)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2137..2361
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2407..2835
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2494..2513)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2704..2725
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2844..2864)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2849..3178
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3029..3164
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3091..3110
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3245..4039
                     /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(3299..3318)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3909..3928
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4213..4334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4250..4269)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4304..4323
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4383..4399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4383..4399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4396..4418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4407..4423
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4431..4437,4438..4455,4456..4461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4476..4497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4614..4631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4785..5373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4865..4884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5544..6335,6336..6404))
                     /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     6167..6186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6405..6509)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6584..6603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gccaccatgg cttctaactt tactcagttc gttctcgtcg acaatggcgg aactggcgac
      721 gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg aatggatcag ctctaactcg
      781 cgatcacagg cttacaaagt aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa
      841 tacaccatca aagtcgaggt gcctaaagtg gcaacccaga ctgttggtgg tgaagagctt
      901 cctgtagccg gatggagatc ttacttaaat atggaactaa ccattccaat tttcgccacg
      961 aattccgact gcgagcttat tgttaaggca atgcaaggtc tcctaaaaga tggaaacccg
     1021 attccctcgg ccatcgcggc caactccggc atctacggag gtggaggtgg atctggtgga
     1081 tctcctccac atatggctta cccatacgat gttccagatt acgctcctcc aaagcttgga
     1141 tccgaattcc cgggtcgact cgagcccatt gacaaacctt cagattctct cagtataggg
     1201 aacggtgata attcccagca gatatctaac agtgatacgc cttcaccacc acctggtttg
     1261 tcaaaatcca atccagtcat ccccatcagt tcatccaatc acagtgcacg gtcccctttt
     1321 gaaggggcag taacagagtc acagtcgtta ttctcagaca attttcgcca tcccaaccct
     1381 atcccaagtg ggcttcctcc tttccccagc tccccacaga catccagtga ctggcctaca
     1441 gcaccagaac cacagagcct cttcacatca gaaacaatcc cagtatcatc ctctacagac
     1501 tggcaagcag cttttggctt tggttcttct aaacaaccag aggatgactt gggttttgat
     1561 cccttcgatg tcactcgaaa agccttagca gacctgattg agaaggaact gtccgttcaa
     1621 gaccaacctt ccctttcgcc cacatctctt cagaactcct cttcacacac tacaaccgcc
     1681 aaaggtccag gctctggatt cctgcatcct gctgcagcta caaatgccaa ttctctcaat
     1741 agtacctttt cagtcttgcc ccagaggttc cctcaatttc agcagcaccg agcggtttat
     1801 aattcattca gttttccagg ccaggcagcc cgctatcctt ggatggcctt tccacgcaat
     1861 agcatcatgc acttgaacca cacagcaaac cccacctcaa atagtaattt cttggacttg
     1921 aatctcccgc cacagcacaa cacaggtctg ggagggatcc ctgtagcagg ggaagaagag
     1981 gtgaaggttt cgaccatgcc actgtcaacc tcttcccatt cattacaaca aggacagcag
     2041 cctacaagtc tccacactac tgtggcctaa gcggccgcct agagggccct attctatagt
     2101 gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     2161 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2221 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2281 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2341 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2401 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2461 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2521 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2581 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2641 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2701 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2761 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2821 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2881 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2941 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3001 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3061 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3121 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3181 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3241 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3301 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3361 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3421 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3481 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3541 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3601 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3661 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3721 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3781 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3841 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3901 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3961 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4021 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4081 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4141 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4201 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4261 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4321 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4381 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4441 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4501 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4561 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4621 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4681 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4741 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4801 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4861 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4921 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4981 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5041 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5101 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5161 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5221 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5281 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5341 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5401 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5461 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5521 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5581 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5641 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5701 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5761 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5821 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5881 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5941 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6001 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6061 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6121 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6181 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6241 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6301 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6361 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6421 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6481 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6541 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     6601 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     6661 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     6721 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtt
//
