LOCUS       sequence-290080-        6970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot3-del632-654.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  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 6970)
  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-del632-654_T
FEATURES             Location/Qualifiers
     source          1..6970
                     /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    3758..3879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3795..3814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3849..3868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3886..4341)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4023..4044)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4235..4254
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4368..4472
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4474..4831
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4474..4665
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4497..4517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4682..4817
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4866..5660
                     /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             4866..5660
                     /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(4920..4939)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5530..5549
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5848..5867)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5892..5939
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6268..6856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6757..6776
                     /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 cccctcacca aggagcagct ctatcagcag gccatggaag aggccgcctg
     3301 gcaccacatg cctcacccct ctgactctcc acactcggac actgtggaat tctaccagcg
     3361 cctgtcgacc gagacactct tcttcatctt ctactatctg gagggcacta aggcacagta
     3421 tctggcagcc aaggccctaa agaagcagtc atggcgattc cacaccaagt acatgatgtg
     3481 gttccagagg cacgaggagc ccaagaccat cactgacgag tttgagcagg gcacctacat
     3541 ctactttgac tacgagaagt ggggccagcg gaagaaggaa ggcttcacct ttgagtaccg
     3601 ctacctggag gaccgggacc tccagtgagg atccaccgga tctagataac tgatcataat
     3661 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     3721 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     3781 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3841 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta
     3901 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3961 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4021 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4081 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4141 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4201 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4261 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4321 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4381 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4441 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     4501 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4561 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4621 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4681 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4741 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4801 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     4861 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4921 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4981 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     5041 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     5101 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     5161 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5221 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5281 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5341 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     5401 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5461 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5521 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     5581 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5641 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5701 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5761 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5821 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     5881 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     5941 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     6001 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     6061 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     6121 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6181 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6241 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6301 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6361 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6421 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6481 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6541 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6601 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6661 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6721 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6781 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6841 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6901 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6961 cgccatgcat
//
