LOCUS       sequence-289955-        6433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot6-L34EI36E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6433)
  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 6433)
  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-HsNot6-L34EI36E_S
FEATURES             Location/Qualifiers
     source          1..6433
                     /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    3221..3342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3258..3277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3312..3331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3349..3804)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3486..3507)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3698..3717
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3831..3935
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3937..4294
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3937..4128
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3960..3980)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4145..4280
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4329..5123
                     /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             4329..5123
                     /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(4383..4402)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4993..5012
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5311..5330)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5355..5402
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5731..6319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6220..6239
                     /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 ctatgcccaa agaaaaatac gagccccctg accctcggag
     1441 gatgtataca attatgtctt ctgaggaagc agcaaatgga aagaaatccc actgggcaga
     1501 ggaagaagaa agtggaaaag taagaagctt aagcgcatct ttgtggtcac taactcacct
     1561 gacagctttg catttgagtg acaattccct gtcccgaatt ccttcagaca ttgccaagct
     1621 tcacaatctg gtgtatttgg acctgtcatc taataaaatt cgtagcttac ccgcagaact
     1681 cggaaacatg gtatcactca gggagctcca tttaaataac aacctgttac gagttctacc
     1741 ttttgagctg ggaaaactgt ttcagttgca gactttaggc ctgaaaggaa atccccttac
     1801 ccaggatata ttgaaccttt atcaggaacc agatggaaca agacggctgc tgaactattt
     1861 gcttgataat ttgtcaggta ctgcaaaaag aattacaaca gaacaaccac ctccaaggtc
     1921 ttggattatg ttacaagaac cagataggac aaggccaact gccttgtttt ctgtcatgtg
     1981 ctataatgtt ctttgtgata aatatgcgac ccggcagtta tacggctact gtccatcatg
     2041 ggcgctaaac tgggactaca ggaaaaaggc cattattcaa gaaatcttga gctgcaatgc
     2101 tgatatcgta agtcttcagg aggttgaaac ggaacagtat tacagttttt ttctggtaga
     2161 gctgaaagaa cgtggctata atggattctt cagtcctaag tctagagcta ggacaatgtc
     2221 agaacaagaa aggaaacatg ttgatggctg tgcaatattc ttcaagacag aaaaatttac
     2281 tttggttcag aaacacactg ttgaatttaa tcagctagcc atggcaaatt ctgaggggtc
     2341 tgaagctatg ctgaacagag tcatgacaaa agataacatt ggggttgcag tactgctaga
     2401 acttcggaag gaatcgattg aaatgccgtc tggaaagcca catcttggaa cagaaaaaca
     2461 acttattctt gtggctaacg cccacatgca ttgggaccct gaatactctg atgtgaagtt
     2521 ggtacaaact atgatgttcc tctcagaagt gaagaacatt attgataaag cctctcgcaa
     2581 cctcaaatcc agtgttttgg gagaatttgg aactattcca cttgtgttat gtgcagatct
     2641 taattctttg ccagactctg gtgttgtaga atatttgagc acaggtggag tagaaacaaa
     2701 tcacaaagac tttaaggagt tgaggtataa tgaaagtctc acaaacttca gctgtcatgg
     2761 gaagaatgga accaccaatg gaaggatcac tcatggtttc aagttacaga gtgcctatga
     2821 gagtggcctg atgccttaca cgaattacac atttgatttc aagggtataa tagactacat
     2881 tttctattct aaacctcagc tgaacacctt aggcatcctg ggccctctgg accaccactg
     2941 gctggttgag aataacatca gtggctgccc gcaccccctc atcccctctg accacttctc
     3001 actttttgca caactggagc tcttactgcc tttcctgccc caagtcaacg gcatccacct
     3061 tcctggcagg aggtagggta ccgcgggccc gggatccacc ggatctagat aactgatcat
     3121 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3181 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3241 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3301 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg
     3361 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3421 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3481 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3541 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3601 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3661 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3721 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3781 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3841 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3901 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3961 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4021 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     4081 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4141 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4201 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4261 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     4321 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4381 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4441 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4501 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4561 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4621 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4681 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4741 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4801 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4861 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4921 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4981 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5041 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5101 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5161 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5221 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5281 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     5341 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     5401 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     5461 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     5521 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     5581 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5641 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5701 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5761 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5821 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5881 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5941 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6001 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6061 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6421 taccgccatg cat
//
