LOCUS       sequence-289246-        5475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot2_344-540.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5475)
  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 5475)
  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_344-540_K
FEATURES             Location/Qualifiers
     source          1..5475
                     /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    2263..2384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2300..2319)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2354..2373
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2391..2846)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2528..2549)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2740..2759
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2873..2977
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2979..3336
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2979..3170
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3002..3022)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3187..3322
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3371..4165
                     /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             3371..4165
                     /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(3425..3444)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4035..4054
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4353..4372)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4397..4444
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4773..5361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5262..5281
                     /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 gtctggcaag acaacttcaa gtacagatgg
     1441 acccaaattc cctggagata aaagttcaac aacacaaaat aataaccagc agaaaaaagg
     1501 gatccaggtg ttacctgatg gtcgggttac taacattcct caagggatgg tgacggacca
     1561 atttggaatg attggcctgt taacatttat cagggcagca gagacagacc caggaatggt
     1621 acatcttgca ttaggaagtg acttaacaac attaggcctc aatctgaact ctcctgaaaa
     1681 tctctacccc aaatttgcgt caccctgggc atcttcacct tgtcgacctc aagacataga
     1741 cttccatgtt ccatctgagt acttaacgaa cattcacatt agggataagc tggctgcaat
     1801 aaaacttggc cgatatggtg aagaccttct cttctatctc tattacatga atggaggaga
     1861 cgtattacaa cttttagctg cagtggagct ttttaaccgt gattggagat accacaaaga
     1921 agaacgagta tggattacca gggcaccagg catggagcca acaatgaaaa ccaataccta
     1981 tgagagggga acatattact tctttgactg tcttaactgg aggaaagtag ctaaggagtt
     2041 ccatctggaa tatgacaaat tagaagaacg gcctcacctg ccatccacct tcaactacaa
     2101 ccctgctcag caagccttct aaccgcgggc ccgggatcca ccggatctag ataactgatc
     2161 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2221 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     2401 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2461 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2521 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2581 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2641 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2701 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2761 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2821 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2941 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     3001 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3061 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3121 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3181 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3241 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3301 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     3361 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3421 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3481 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3541 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3601 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3661 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3721 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3781 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3841 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3901 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3961 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4021 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4081 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4141 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4201 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4261 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4321 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     4381 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     4441 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     4501 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     4561 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     4621 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4681 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4741 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4801 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4861 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4921 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4981 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5041 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5101 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5161 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5221 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5281 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5341 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5401 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5461 attaccgcca tgcat
//
