LOCUS       sequence-289950-        6955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot3-del607-634-F673E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6955)
  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 6955)
  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-del607-634-F673E_S
FEATURES             Location/Qualifiers
     source          1..6955
                     /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    3743..3864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3780..3799)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3834..3853
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3871..4326)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4008..4029)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4220..4239
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4353..4457
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4459..4816
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4459..4650
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4482..4502)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4667..4802
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4851..5645
                     /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             4851..5645
                     /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(4905..4924)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5515..5534
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5833..5852)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5877..5924
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6253..6841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6742..6761
                     /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 cagcggaggt 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 ccccggcagt acctcccccg gaacccctgt ccgacgcccc cctaccacca
     3301 ccagatgcca cccccacact cggacactgt ggaattctac cagcgcctgt cgaccgagac
     3361 actcttcgaa atcttctact atctggaggg cactaaggca cagtatctgg cagccaaggc
     3421 cctaaagaag cagtcatggc gattccacac caagtacatg atgtggttcc agaggcacga
     3481 ggagcccaag accatcactg acgagtttga gcagggcacc tacatctact ttgactacga
     3541 gaagtggggc cagcggaaga aggaaggctt cacctttgag taccgctacc tggaggaccg
     3601 ggacctccag tgaggatcca ccggatctag ataactgatc ataatcagcc ataccacatt
     3661 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     3721 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     3781 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3841 gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat
     3901 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3961 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4021 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4081 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4141 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4201 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4261 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4321 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4381 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4441 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     4501 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4561 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4621 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4681 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4741 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4801 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4861 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4921 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4981 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     5041 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     5101 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5161 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5221 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5281 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5341 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     5401 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5461 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5521 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     5581 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     5641 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     5701 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5761 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     5821 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     5881 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     5941 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     6001 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     6061 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     6121 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6181 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6241 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6301 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6361 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6421 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6481 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6541 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6601 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6661 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6721 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6781 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6841 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6901 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
