LOCUS       sequence-289946-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot2-del380-404-W507E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  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 6309)
  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-del380-404-W507E_S
FEATURES             Location/Qualifiers
     source          1..6309
                     /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    3097..3218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3134..3153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3188..3207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3225..3680)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3362..3383)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3574..3593
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3707..3811
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3813..4170
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3813..4004
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3836..3856)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4021..4156
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4205..4999
                     /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             4205..4999
                     /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(4259..4278)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4869..4888
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5187..5206)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5231..5278
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5607..6195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6096..6115
                     /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 taggatcttc accttgtcga cctcaagaca tagacttcca
     2581 tgttccatct gagtacttaa cgaacattca cattagggat aagctggctg caataaaact
     2641 tggccgatat ggtgaagacc ttctcttcta tctctattac atgaatggag gagacgtatt
     2701 acaactttta gctgcagtgg agctttttaa ccgtgattgg agataccaca aagaagaacg
     2761 agtatggatt accagggcac caggcatgga gccaacaatg aaaaccaata cctatgagag
     2821 gggaacatat tacttctttg actgtcttaa cgagaggaaa gtagctaagg agttccatct
     2881 ggaatatgac aaattagaag aacggcctca cctgccatcc accttcaact acaaccctgc
     2941 tcagcaagcc ttctaaccgc gggcccggga tccaccggat ctagataact gatcataatc
     3001 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     3061 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     3121 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3181 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     3241 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     3301 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     3361 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     3421 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     3481 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     3541 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3601 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3661 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3721 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3781 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     3841 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3901 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3961 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4021 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4081 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4141 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     4201 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4261 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4321 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4381 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4441 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4501 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4561 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4621 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4681 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     4741 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4801 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4861 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4921 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4981 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5041 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5101 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5161 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     5221 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     5281 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     5341 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     5401 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     5461 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5521 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5581 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5641 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5701 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5761 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5821 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5881 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5941 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6001 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6061 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6121 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6181 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6241 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6301 gccatgcat
//
