LOCUS       sequence-289949-        6384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot2-L431AL436AL459A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  AUTHORS   Boland A, Chen Y, Raisch T, Jonas S, Kuzuoglu-Ozturk D, Wohlbold L, 
            Weichenrieder O, Izaurralde E
  TITLE     Structure and assembly of the NOT module of the human CCR4-NOT 
            complex.
  JOURNAL   Nat Struct Mol Biol. 2013 Nov;20(11):1289-97. doi: 
            10.1038/nsmb.2681. Epub 2013 Oct 13.
  PUBMED    24121232
REFERENCE   2  (bases 1 to 6384)
  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"; doi: "10.1038/nsmb.2681";
            journalName: "Nat Struct Mol Biol"; date: "2013-11"; volume: "20"; 
            issue: "11"; pages: "1289-97"
COMMENT     Sequence Label: pT7-EGFP-C1-HsNot2-L431AL436AL459A_S
FEATURES             Location/Qualifiers
     source          1..6384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     613..632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      659..668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(710..731)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(971..990)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1318..1339
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3172..3293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3209..3228)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3263..3282
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3300..3755)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3437..3458)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3649..3668
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3782..3886
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3888..4245
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3888..4079
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3911..3931)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4096..4231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4280..5074
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4280..5074
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4334..4353)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4944..4963
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5262..5281)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5306..5353
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5682..6270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6171..6190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      601 gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcgc taccggtcgc
      661 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      721 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      781 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      841 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      901 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      961 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1021 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1081 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1141 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1201 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1261 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1321 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1381 gtccggactc agatctcgag ctcaagcttc ggtgaggact gatggacata cattatctga
     1441 gaaaagaaac taccaggtga caaacagcat gtttggtgct tcaagaaaga agtttgtaga
     1501 gggggtcgac agtgactacc atgacgaaaa catgtactac agccagtctt ctatgtttcc
     1561 acatcggtca gaaaaagata tgctggcatc accatctaca tcaggtcagc tgtctcagtt
     1621 tggggcaagt ttatacgggc aacaaagtgc actaggcctt ccaatgaggg ggatgagcaa
     1681 caatacccct cagttaaatc gcagcttatc acaaggcact cagttaccga gccacgtcac
     1741 gccaacaaca ggggtaccaa caatgtcact tcacacgcct ccatctccaa gcaggggtat
     1801 tttgcctatg aatcctagga atatgatgaa ccactcccag gttggtcagg gcattggaat
     1861 tcctagcagg acaaatagca tgagcagttc agggttaggt agccccaaca gaagctcgcc
     1921 aagcataata tgtatgccaa agcagcagcc ttctcgacag ccttttactg tgaacagtat
     1981 gtctggattt ggaatgaaca ggaatcaggc atttggaatg aataactcct tatcaagtaa
     2041 catttttaat ggaacagacg gaagtgaaaa tgtgacagga ttggaccttt cagatttccc
     2101 agcattagca gaccgaaaca ggagggaagg aagtggtaac ccaactccat taataaaccc
     2161 cttggctgga agagctcctt atgttggaat ggtaacaaaa ccagcaaatg aacaatccca
     2221 ggacttctca atacacaatg aagattttcc agcattacca ggctccagct ataaagatcc
     2281 aacatcaagt aatgatgaca gtaaatctaa tttgaataca tctggcaaga caacttcaag
     2341 tacagatgga cccaaattcc ctggagataa aagttcaaca acacaaaata ataaccagca
     2401 gaaaaaaggg atccaggtgt tacctgatgg tcgggttact aacattcctc aagggatggt
     2461 gacggaccaa tttggaatga ttggcctgtt aacatttatc agggcagcag agacagaccc
     2521 aggaatggta catcttgcat taggaagtga cttaacaaca ttaggcctca atctgaactc
     2581 tcctgaaaat ctctacccca aatttgcgtc accctgggca tcttcacctt gtcgacctca
     2641 agacatagac ttccatgttc catctgagta cttaacgaac attcacatta gggataaggc
     2701 cgctgcaata aaagccggcc gatatggtga agaccttctc ttctatctct attacatgaa
     2761 tggaggagac gtattacaac ttgcagctgc agtggagctt tttaaccgtg attggagata
     2821 ccacaaagaa gaacgagtat ggattaccag ggcaccaggc atggagccaa caatgaaaac
     2881 caatacctat gagaggggaa catattactt ctttgactgt cttaactgga ggaaagtagc
     2941 taaggagttc catctggaat atgacaaatt agaagaacgg cctcacctgc catccacctt
     3001 caactacaac cctgctcagc aagccttcta accgcgggcc cgggatccac cggatctaga
     3061 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3121 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     3181 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3241 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta
     3301 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3361 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3421 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3481 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3541 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3601 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3661 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3721 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     3781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3841 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     3901 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3961 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     4021 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     4081 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4141 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4201 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     4261 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4321 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4381 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4441 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4501 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4561 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4621 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4681 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4741 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4801 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4861 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4921 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4981 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5041 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5101 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5161 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5221 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     5281 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     5341 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     5401 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     5461 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     5521 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5581 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5641 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5701 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5761 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5821 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5881 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5941 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6001 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6061 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6121 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6181 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6241 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6301 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6361 gataaccgta ttaccgccat gcat
//
