LOCUS       sequence-290076-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of DmNot2-del344-380.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  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 6273)
  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-del344-380_T
FEATURES             Location/Qualifiers
     source          1..6273
                     /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    3061..3182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3098..3117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3152..3171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3189..3644)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3326..3347)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3538..3557
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3671..3775
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3777..4134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3777..3968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3800..3820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3985..4120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4169..4963
                     /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             4169..4963
                     /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(4223..4242)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4833..4852
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5151..5170)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5195..5242
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5571..6159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6060..6079
                     /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 tcgggttgac ttaacaacat taggcctcaa
     2461 tctgaactct cctgaaaatc tctaccccaa atttgcgtca ccctgggcat cttcaccttg
     2521 tcgacctcaa gacatagact tccatgttcc atctgagtac ttaacgaaca ttcacattag
     2581 ggataagctg gctgcaataa aacttggccg atatggtgaa gaccttctct tctatctcta
     2641 ttacatgaat ggaggagacg tattacaact tttagctgca gtggagcttt ttaaccgtga
     2701 ttggagatac cacaaagaag aacgagtatg gattaccagg gcaccaggca tggagccaac
     2761 aatgaaaacc aatacctatg agaggggaac atattacttc tttgactgtc ttaactggag
     2821 gaaagtagct aaggagttcc atctggaata tgacaaatta gaagaacggc ctcacctgcc
     2881 atccaccttc aactacaacc ctgctcagca agccttctaa ccgcgggccc gggatccacc
     2941 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     3001 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     3061 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3121 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3181 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3241 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3301 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3361 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3421 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3481 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3541 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3601 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     3661 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3721 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     3781 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3901 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3961 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4021 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4081 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     4141 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4201 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4261 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4321 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     4381 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4441 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4501 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4561 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4621 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4681 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4741 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4801 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4861 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4921 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4981 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5041 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5101 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     5161 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     5221 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     5281 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     5341 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     5401 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     5461 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5521 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5581 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5641 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5701 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     5761 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5821 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5881 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5941 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6001 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6061 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6121 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6181 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6241 gattctgtgg ataaccgtat taccgccatg cat
//
