LOCUS       sequence-290458-        4646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNanos2-W9E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4646)
  AUTHORS   Bhandari D, Raisch T, Weichenrieder O, Jonas S, Izaurralde E
  TITLE     Structural basis for the Nanos-mediated recruitment of the CCR4-NOT 
            complex and translational repression.
  JOURNAL   Genes Dev. 2014 Apr 15;28(8):888-901. doi: 10.1101/gad.237289.113.
  PUBMED    24736845
REFERENCE   2  (bases 1 to 4646)
  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: 
            "10.1101/gad.237289"; journalName: "Genes Dev"; date: "2014-04-15- 
            15"; volume: "28"; issue: "8"; pages: "888-901"
COMMENT     Sequence Label: pT7-V5-SBP-C1-HsNanos2-W9E_Y
FEATURES             Location/Qualifiers
     source          1..4646
                     /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             668..709
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             731..844
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWGGGHVVEGLAGELEQLRARLEHHPQGQREP"
     polyA_signal    1434..1555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1471..1490)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1525..1544
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1562..2017)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1699..1720)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1911..1930
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2044..2148
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2150..2507
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2150..2341
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2173..2193)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2358..2493
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2542..3336
                     /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             2542..3336
                     /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(2596..2615)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3206..3225
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3524..3543)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3568..3615
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3944..4532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4433..4452
                     /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 caccatgggt aagcctatcc ctaaccctct cctcggtctc gattctacgc ccggaagcgg
      721 tagcggtacg atggacgaga agaccaccgg ctgggggggc ggccacgtgg tggagggcct
      781 ggccggcgag ctggagcagc tgcgggccag gctggagcac caccctcagg gccagcggga
      841 gcccagatct cgagctatgc agctgccccc attcgacatg gagaaggact acttcaacct
      901 gagccaggtc gtgtgggccc tgatcgccag cagaggccag agactggaaa cccaggaaat
      961 cgaggaaccc agccctggcc ctccactggg acaggatcag ggactgggag cccctggcgc
     1021 taatggcgga ctgggcaccc tgtgcaattt ctgcaagcac aacggcgaga gccggcacgt
     1081 gtacagcagc caccagctga aaacccccga cggcgtggtc gtgtgcccca tcctgagaca
     1141 ctacgtgtgc cctgtgtgcg gcgccactgg ggatcaggcc cataccctga agtactgccc
     1201 cctgaatggc ggccagcagt ccctgtacag aagaagcggc agaaacagcg ctgggcggag
     1261 agtgaagaga taagaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta
     1321 gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     1381 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     1441 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1501 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg
     1561 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     1621 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     1681 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     1741 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     1801 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     1861 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     1921 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     1981 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     2041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     2161 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2221 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2281 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2341 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2401 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2461 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     2521 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2581 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2641 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2701 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     2761 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2821 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2881 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2941 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3001 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3061 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3121 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3181 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3241 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3301 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3361 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3421 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3481 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     3541 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     3601 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     3661 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     3721 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     3781 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3841 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3901 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3961 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4021 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4081 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4141 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4201 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4261 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4321 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4381 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4441 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4501 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4561 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4621 tggataaccg tattaccgcc atgcat
//
