LOCUS       sequence-289828-        5570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot7-T142Y.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5570)
  AUTHORS   Petit AP, Wohlbold L, Bawankar P, Huntzinger E, Schmidt S, 
            Izaurralde E, Weichenrieder O
  TITLE     The structural basis for the interaction between the CAF1 nuclease 
            and the NOT1 scaffold of the human CCR4-NOT deadenylase complex.
  JOURNAL   Nucleic Acids Res. 2012 Nov;40(21):11058-72. doi: 
            10.1093/nar/gks883. Epub 2012 Sep 12.
  PUBMED    22977175
REFERENCE   2  (bases 1 to 5570)
  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: 
            "10.1093/nar/gks883"; journalName: "Nucleic Acids Res"; date: 
            "2012-11"; volume: "40"; issue: "21"; pages: "11058-72"
COMMENT     Sequence Label: pT7-EGFP-C1-HsNot7-T142Y_R
FEATURES             Location/Qualifiers
     source          1..5570
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2358..2479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2395..2414)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2449..2468
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2486..2941)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2623..2644)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2835..2854
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2968..3072
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3074..3431
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3074..3265
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3097..3117)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3282..3417
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3466..4260
                     /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             3466..4260
                     /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(3520..3539)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4130..4149
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4448..4467)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4492..4539
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4868..5456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5357..5376
                     /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 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctatgcca gcggcaactg tagatcatag ccaaagaatt
     1381 tgtgaagttt gggcttgcaa cttggatgaa gagatgaaga aaattcgtca agttatccga
     1441 aaatataatt acgttgctat ggacaccgag tttccaggtg tggttgcaag acccattgga
     1501 gaattcagga gcaatgctga ctatcaatac caactattgc ggtgtaatgt agacttgtta
     1561 aagataattc agctaggact gacatttatg aatgagcaag gagaataccc tccaggaact
     1621 tcaacttggc agtttaattt taaatttaat ttgacggagg acatgtatgc ccaggactct
     1681 atagagctac taacaacatc tggtatccag tttaaaaaac atgaggagga aggaattgaa
     1741 acccagtact ttgcagaact tcttatgtat tctggagtgg tcctctgtga aggggtcaaa
     1801 tggttgtcat ttcatagcgg ttacgacttt ggctacttaa tcaaaatcct aaccaactct
     1861 aacttgcctg aagaagaact tgacttcttt gagatccttc gattgttttt tcctgtcatt
     1921 tatgatgtga agtacctcat gaagagctgc aaaaatctca aaggtggatt acaggaggtg
     1981 gcagaacagt tagagctgga acggatagga ccacaacatc aggcaggatc tgattcattg
     2041 ctcacaggaa tggccttttt caaaatgaga gaaatgttct ttgaagatca tattgatgat
     2101 gccaaatatt gtggtcattt gtatggcctt ggttctggtt catcctatgt acagaatggc
     2161 acagggaatg catatgaaga ggaagccaac aagcagtcat gactgcagtc gacggtaccg
     2221 cgggcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     2281 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2341 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2401 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2461 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     2521 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2581 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2641 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2701 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     2761 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2821 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     2881 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2941 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3001 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3061 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3121 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3181 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3241 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3301 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3361 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3421 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3481 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3541 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3601 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     3661 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3721 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3781 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3841 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3901 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3961 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4021 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4081 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4141 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4201 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4261 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4321 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4381 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     4441 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     4501 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4561 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     4621 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     4681 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     4741 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4801 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4861 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4921 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4981 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5041 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5101 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5161 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5221 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5281 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5341 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5401 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5461 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5521 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
