LOCUS       sequence-289954-        7039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot3-F727A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7039)
  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 7039)
  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-HsNot3-F727A_S
FEATURES             Location/Qualifiers
     source          1..7039
                     /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    3827..3948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3864..3883)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3918..3937
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3955..4410)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4092..4113)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4304..4323
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4437..4541
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4543..4900
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4543..4734
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4566..4586)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4751..4886
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4935..5729
                     /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             4935..5729
                     /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(4989..5008)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5599..5618
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5917..5936)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5961..6008
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6337..6925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6826..6845
                     /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 gaattctgca gtcgacggta ccgcgggcgc
     1441 ggacaagcgc aaactccaag gtgagattga tcgctgcctc aagaaggtgt ccgagggcgt
     1501 ggagcagttt gaagatattt ggcagaagct ccacaatgca gccaacgcga accagaaaga
     1561 aaagtatgag gctgacctaa agaaggagat taagaagcta caacggctga gggaccaaat
     1621 caagacatgg gtagcgtcca acgagatcaa ggacaagagg cagcttatag acaaccgcaa
     1681 gctcattgag acgcaaatgg aacggttcaa agttgtggaa cgagagacca aaaccaaagc
     1741 ttacagcaaa gagggcctgg gcctggccca gaaggtagat cctgcccaga aggagaagga
     1801 agaggttggc cagtggctca cgaataccat cgacacgctc aacatgcagg tggaccagtt
     1861 tgagagtgaa gtggagtcac tgtcagtgca gacacgcaag aagaagggcg acaaggataa
     1921 gcaggaccgg attgagggct tgaagcggca catcgagaag caccgctacc acgtgcgcat
     1981 gctagagacc atcctgcgca tgctggacaa tgactccatc ctcgttgacg ccatccgcaa
     2041 gatcaaggac gacgttgagt actatgttga ctcatcccag gaccccgact tcgaggagaa
     2101 cgagtttctc tacgatgacc tggacctcga ggacattcca caggcgctgg tcgccacctc
     2161 cccccccagc cacagccaca tggaggatga gatcttcaac cagtccagca gcacgcccac
     2221 ctcaaccacc tccagctctc ccatcccgcc cagcccagcc aactgtacca cggaaaactc
     2281 tgaagatgat aagaagaggg gacgttccac agacagtgaa gtcagccagt ctccagccaa
     2341 aaacggctcc aagcctgtcc acagcaacca gcaccctcag tccccagctg tgccgcccac
     2401 ctacccctcc ggccccccgc ctgctgcctc tgccttgagc accactcctg gcaacaatgg
     2461 ggtccccgcc cccgcagcac ccccaagtgc cctgggcccc aaggccagtc cagctcccag
     2521 ccacaactcg ggcacccctg ctccctatgc ccaggctgtg gccccaccag ctcccagtgg
     2581 gcccagcacg acccagcccc ggccccccag cgtccagcct agcggaggcg gaggcggcgg
     2641 cagcggaggc ggagggagca gcagcagtag taacagcagt gccggtggag gggctggcaa
     2701 gcagaatggc gccaccagtt acagctcagt tgtggcagac agcccggcag aggtggcttt
     2761 gagcagcagt gggggcaaca atgccagcag ccaggccttg ggcccccctt ccggccccca
     2821 caacccacct cccagcacct cgaaggaacc cagtgcggca gccccaacgg gggctggggg
     2881 cgtggcccca ggctcaggga acaactcagg gggacccagc ctcctggtgc cactgcctgt
     2941 gaatcctccc agctccccaa cgcccagctt cagtgatgcc aaggcagccg gtgccctgct
     3001 caatgggcct ccacagttca gcaccgcccc agaaatcaag gcccctgagc ctctgagctc
     3061 cttgaagtcc atggcggaac gggcagccat cagctctggc attgaggacc ctgtgccaac
     3121 gctgcacctg accgagcgag acatcatcct gagcagtaca tcagcacctc cggcctcagc
     3181 ccagccgccc ctgcagctgt cagaggtgaa cataccgctg tcgctgggtg tctgtccact
     3241 gggccctgtg cccctcacca aggagcagct ctatcagcag gccatggaag aggccgcctg
     3301 gcaccacatg cctcacccct ctgactctga gcgtattcgg cagtacctcc cccggaaccc
     3361 ctgtccgacg cccccctacc accaccagat gccaccccca cactcggaca ctgtggaatt
     3421 ctaccagcgc ctgtcgaccg agacactctt cttcatcttc tactatctgg agggcactaa
     3481 ggcacagtat ctggcagcca aggccctaaa gaagcagtca tggcgattcc acaccaagta
     3541 catgatgtgg ttccagaggc acgaggagcc caagaccatc actgacgagt ttgagcaggg
     3601 cacctacatc tacgcagact acgagaagtg gggccagcgg aagaaggaag gcttcacctt
     3661 tgagtaccgc tacctggagg accgggacct ccagtgagga tccaccggat ctagataact
     3721 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3781 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3841 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3901 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg
     3961 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4021 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4081 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4141 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4201 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4261 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4321 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4381 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     4441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4501 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     4561 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4621 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4681 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4741 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4801 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4861 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     4921 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4981 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     5041 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     5101 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     5161 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     5221 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     5281 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     5341 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     5401 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     5461 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     5521 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5581 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5641 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5701 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5761 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5821 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5881 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     5941 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     6001 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     6061 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     6121 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     6181 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6241 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6301 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6361 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6421 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6481 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6541 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6601 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6661 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6721 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6781 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6841 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6901 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6961 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7021 ccgtattacc gccatgcat
//
