LOCUS       sequence-496741-        6384 bp    DNA     circular SYN 08-JAN-2026
DEFINITION  Expresses FLAG, His tagged Siwi in silkworm cell line BmN4 or insect
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  AUTHORS   Kawaoka S, Hayashi N, Suzuki Y, Abe H, Sugano S, Tomari Y, Shimada 
            T, Katsuma S
  TITLE     The Bombyx ovary-derived cell line endogenously expresses 
            PIWI/PIWI-interacting RNA complexes.
  JOURNAL   RNA. 2009 Jul;15(7):1258-64. doi: 10.1261/rna.1452209. Epub 2009 May
            21.
  PUBMED    19460866
REFERENCE   2  (bases 1 to 6384)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 8, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1261/rna.1452209"; journalName: "RNA"; date: "2009-07"; volume: 
            "15"; issue: "7"; pages: "1258-64"
COMMENT     Sequence Label: pIZ-Flag6His-Siwi
FEATURES             Location/Qualifiers
     source          1..6384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..548
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 promoter
                     /note="strong constitutive baculovirus promoter for insect 
                     cell expression"
     CDS             584..607
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             608..625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4160..4201
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4211..4228
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4246..4375
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     rep_origin      complement(4503..5091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4583..4602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5163..5710
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 promoter
                     /note="strong constitutive baculovirus promoter for insect 
                     cell expression"
     promoter        5746..5793
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5785..5803
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5807..5816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5813..6187
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    join(6251..6384,1)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6288..6307)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6342..6361
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 catgatgata aacaatgtat ggtgctaatg ttgcttcaac aacaattctg ttgaactgtg
       61 ttttcatgtt tgccaacaag cacctttata ctcggtggcc tccccaccac caactttttt
      121 gcactgcaaa aaaacacgct tttgcacgcg ggcccataca tagtacaaac tctacgtttc
      181 gtagactatt ttacataaat agtctacacc gttgtatacg ctccaaatac actaccacac
      241 attgaacctt tttgcagtgc aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc
      301 gggtcgcgtc ctgtcacgta cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt
      361 gacaggacgc cagcttcctg tgttgctaac cgcagccgga cgcaactcct tatcggaaca
      421 ggacgcgcct ccatatcagc cgcgcgttat ctcatgcgcg tgaccggaca cgaggcgccc
      481 gtcccgctta tcgcgcctat aaatacagcc cgcaacgatc tggtaaacac agttgaacag
      541 catctgttcg aatttaaagc ttggtaccga gctcggatcc atggattaca aggatgacga
      601 cgataagcat catcatcatc atcatatgtc agaaccgaga ggtagaggac gagctcgagg
      661 acgcgctggt cgtggtggtg atggaggcgg accagcacca cgacgtccag gggaacaagc
      721 aggcccatca caacaatcta tgccacctgg tcctagacct caacctccat ctggctgggg
      781 accacaatcc agtgttcctc cagtaagagc tggtgtcccg acaccaacag cacaagcggg
      841 tcgcgcctca caccgtgtta ctcccactac acatgaacat ccaggagata tcgatgtgca
      901 acaacgcatg caaaaattag aactgggacc ccattcatct ggaggtggtg atgcatcatc
      961 cgtagttggc cgtggctctc gacgaggggg tggaagagtc cttcctgaaa ctatttccat
     1021 cttgcgcaca cgtccagaag ctgtaacatc taagaaaggt acttcgggta ctcccctgga
     1081 tctgcttgcc aactatttta ctgttgaaac aactcccaaa tggggcctgt atcaatatca
     1141 cgtcgatatt tctccagaag aagacagcac tggagtccga aaggcactga tgcgtgttca
     1201 cagtaaaaca ctcggaggat atctctttga tggcactgtg ttgtacactg taaatcgtct
     1261 gcatcctgat cccatggagc tatactctga tcgtaaaaca gataacgaac gtatgcgaat
     1321 cttaattaag ctgacctgcg aagttagccc tggagactat cactatatac aaatctttaa
     1381 catcattata aggaaatgct ttaatttatt gaagctacag cttatgggac gggattattt
     1441 tgaccctgaa gcaaagattg atataccaga attcaagctt caaatttggc caggatataa
     1501 aacgacaatc aatcaatatg aagatcgttt gttgttggtc actgaaatcg cccataaggt
     1561 gcttcgcatg gacactgttc ttcaaatgtt gagtgaatat gccgcaacaa aaggcaataa
     1621 ttacaaaaaa atatttttag aagatgttgt tggaaaaatt gtaatgactg actacaataa
     1681 aagaacttac agagttgacg atgtagcttg gaacgtgtcg ccgaagtcca catttaaaat
     1741 gcgtgacgaa aacattacct atattgaata ctattataag aaatacaatc tacgcattca
     1801 agatccgggt caaccgcttc tgatctcgcg gtcgaagccg cgtgagattc gtgcgggtct
     1861 gcctgagttg atatatctgg tcccagaact gtgccgtcag accggcctct ccgacgagat
     1921 gcgagctaat ttcaagctga tgcgctcatt ggacgtccat acaaagattg ggcccgacaa
     1981 gcgcattgaa aagcttaata atttcaacag gaggttcaca agcacacctg aagttgttga
     2041 agaactggcc acatggtcgt tgaaactatc taaggaactg gtaaaaataa agggtcgcca
     2101 acttccacct gaaaatataa tacaagcaaa taatgtcaaa taccctgcag gagacaccac
     2161 tgagggctgg acgcgagata tgagatcgaa gcatctcctg gctattgccc aactaaactc
     2221 ttgggtagtg atcaccccag aaagacaacg ccgtgacaca gagtcattca ttgatctcat
     2281 aataaaaact ggcggaggtg ttggttttcg aatgagaagc cctgacttag ttgtgattcg
     2341 tcatgacggt ccgatagaat acgctaatat gtgtgaagaa gtgattgcaa ggaaaaatcc
     2401 tgccctgata ctgtgcgttc tggctaggaa ttatgctgat aggtatgaag ccataaaaaa
     2461 gaaatgcact gtagaccgag ccgtgccgac gcaggtagtc tgcgctcgta acatgagtag
     2521 caagtcggct atgtcaattg ctacaaaagt tgctattcaa ataaattgta agcttggggg
     2581 ttctccgtgg acagtagata ttccactgcc gtcactgatg gtggtcgggt acgacgtgtg
     2641 ccacgacact cgttcgaaag aaaagagctt cggggccttc gtagccaccc tcgacaaaca
     2701 gatgactcag tactattcaa tcgtcaatgc gcacacttcc ggagaagaac tcagctctca
     2761 catgggtttc aatatcgctt ccgccgtcaa aaaattccgc gagaaaaacg ggacctaccc
     2821 agcccgaatc ttcatttacc gcgacggtgt cggtgatggt caaatcccat acgtgcacag
     2881 ccatgaagtt gctgagataa aaaagaaatt ggcagaaatt tatgcaggag tcgaaataaa
     2941 gttggcattc attattgtgt cgaaacgtat taacacccga atcttcgttc aacgtggtcg
     3001 cagcggagaa aatccacgcc ctggcacagt gattgacgac gtcgtcacct tgcctgaacg
     3061 gtatgatttc tatctggttt cacaaaatgt acgtgaaggt accattgctc ctacctcata
     3121 caatgtgatt gaagatacaa caggactcaa tcctgatcgt atccaacgtt tgacctacaa
     3181 actgacacac ttgtatttca actgctcttc ccaagttcgc gtgccatcgg tctgccaata
     3241 tgcccacaaa cttgcattcc ttgcagctaa cagtctgcac aaccaaccgc attattcatt
     3301 gaatgaaact ttatatttcc tctaagtatt tttaaagaca acctattttt ttctagatta
     3361 agtaagtgta cccaccgaga aactattttc gatgtgcata accatacaaa tcatagaatt
     3421 ctattagaaa tagttcgcaa tcataacgtg agcttaatat tgctcaaagt aattcatttc
     3481 atagtttttt aaaagatata ttaaataaga ctcattctct tggacgtttt tgatgtatga
     3541 ttagtgtggc atagcaagaa gcagctaatg cgcgtagtag tagtacatgt gacctagact
     3601 cgtttttacg ataattaaaa tgtaatgagt gttctagttg tttactacat aagattcatt
     3661 ttatatttga ttgacaattg tcaaattttt ctttttattt tggctacaat gatgttgagt
     3721 ggaaatgcaa acgaataatt attaaattac aagttattca aattgaatca agtgcattct
     3781 tccacgctac aagcagtgat ctgaaatcta gccaacgtat aattagtttt tttttttttt
     3841 tgctctgtta cttaaggctt ttatatcaac gatgttacct cgttttagtt ctaagtttcg
     3901 tgataagttt tgacttacat gcaacatgta gcttttagaa aagatttttg tttttcatgt
     3961 ttttttttat tacaatttca tttttaaaaa aaaaaaaaaa aaagaataaa aaaaaaaaaa
     4021 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4081 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag taccgcggcc gctcgagtct
     4141 agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac
     4201 gcgtaccggt catcatcacc atcaccattg agtttatctg actaaatctt agtttgtatt
     4261 gtcatgtttt aatacaatat gttatgttta aatatgtttt taataaattt tataaaataa
     4321 tttcaacttt tattgtaaca acattgtcca tttacacact cctttcaagc gcgtgggatc
     4381 gatgctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4441 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4681 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4921 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5161 gtcatgatga taaacaatgt atggtgctaa tgttgcttca acaacaattc tgttgaactg
     5221 tgttttcatg tttgccaaca agcaccttta tactcggtgg cctccccacc accaactttt
     5281 ttgcactgca aaaaaacacg cttttgcacg cgggcccata catagtacaa actctacgtt
     5341 tcgtagacta ttttacataa atagtctaca ccgttgtata cgctccaaat acactaccac
     5401 acattgaacc tttttgcagt gcaaaaaagt acgtgtcggc agtcacgtag gccggcctta
     5461 tcgggtcgcg tcctgtcacg tacgaatcac attatcggac cggacgagtg ttgtcttatc
     5521 gtgacaggac gccagcttcc tgtgttgcta accgcagccg gacgcaactc cttatcggaa
     5581 caggacgcgc ctccatatca gccgcgcgtt atctcatgcg cgtgaccgga cacgaggcgc
     5641 ccgtcccgct tatcgcgcct ataaatacag cccgcaacga tctggtaaac acagttgaac
     5701 agcatctgtt cgaattaatt cgggatctgc cgggctgcag cacgtgttga caattaatca
     5761 tcggcatagt atatcggcat agtataatac gactcactat aggagggcca ccatggccaa
     5821 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
     5881 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
     5941 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
     6001 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
     6061 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
     6121 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     6181 ggactgaccg acgccgacca acaccgccgg tccgacgcgg cccgacgggt ccgagggggg
     6241 tcgacctcga aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6301 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6361 caatgtatct tatcatgtct ggat
//
