LOCUS       sequence-290394-        6403 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian Expression of HsNanos3-W7E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6403)
  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 6403)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pcDNA3.1-MS2-HA-HsNanos3-W7E_X
FEATURES             Location/Qualifiers
     source          1..6403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1333..1359
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1959..1977)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1960..1977)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1997..2014)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2003..2227
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2273..2701
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2360..2379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2570..2591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2710..2730)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2715..3044
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2895..3030
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2957..2976
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3111..3905
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3165..3184)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3775..3794
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4079..4200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4116..4135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4170..4189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4249..4265)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4249..4265)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4262..4284)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4273..4289
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4297..4303,4304..4321,4322..4327))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4342..4363
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4480..4497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4651..5236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4731..4750)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5407..6198,6199..6267))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6030..6049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6268..6372)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc
      901 atggcttcta actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact
      961 gtcgccccaa gcaacttcgc taacggggtc gctgaatgga tcagctctaa ctcgcgatca
     1021 caggcttaca aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc
     1081 atcaaagtcg aggtgcctaa agtggcaacc cagactgttg gtggtgaaga gcttcctgta
     1141 gccggatgga gatcttactt aaatatggaa ctaaccattc caattttcgc cacgaattcc
     1201 gactgcgagc ttattgttaa ggcaatgcaa ggtctcctaa aagatggaaa cccgattccc
     1261 tcggccatcg cggccaactc cggcatctac ggaggtggag gtggatctgg tggatctcct
     1321 ccacatatgg cttacccata cgatgttcca gattacgctc ctccaaagct tggatccgaa
     1381 ttcccgggtc gactcgagat gggcaccttc gacctggaga ccgactatct gggactggcc
     1441 catctcgtgc gggccctgtc tggaaaagag ggccccgaga caagactgag cccccagcct
     1501 gagccagagc ccatgctgga acccgaccag aagcggagcc tggaaagctc tcctgccccc
     1561 gagagactgt gcagcttctg caagcacaac ggcgagagcc gggccatcta ccagagccac
     1621 gtgctgaagg atgaggccgg cagagtgctg tgccccatcc tgagagacta cgtgtgccct
     1681 cagtgcggcg ccaccagaga gagagcccac accagaagat tctgccccct gaccggccag
     1741 ggctacacca gcgtgtacag ccacaccacc agaaacagcg ccggcaagaa actcgtgcgg
     1801 cccgacaagg ccaagaccca ggataccgga cacagaagag gcggcggagg cggagctggc
     1861 tttagaggcg ccggaaagtc tgagcccagc cccagctgta gccccagcat gagcacctaa
     1921 tctagagtcg acccgggcgg ccgcctagag ggccctattc tatagtgtca cctaaatgct
     1981 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2041 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2101 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2161 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2221 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2281 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2341 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2401 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2461 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2521 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2581 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2641 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2701 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2761 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2821 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2881 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2941 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3001 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3061 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3121 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3181 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3241 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3301 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3361 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3421 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3481 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3541 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3601 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3661 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3721 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3781 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3841 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3901 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3961 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4021 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4081 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4141 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4201 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4261 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4321 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4381 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4441 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4501 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4561 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4621 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4681 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4741 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4801 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4861 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4921 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4981 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5041 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5101 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5161 cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga ttacgcgcag
     5221 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5281 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5341 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5401 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5461 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5521 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5581 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5641 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5701 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5761 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5821 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5881 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5941 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6001 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6061 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6361 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
