LOCUS       sequence-486815-        6301 bp    DNA     circular SYN 22-SEP-2025
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 6301)
  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 6301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 22, 2025 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..6301
                     /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             4077..4118
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4128..4145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4163..4292
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     rep_origin      complement(4420..5008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4500..4519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5080..5627
                     /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        5663..5710
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5702..5720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5724..5733
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5730..6104
                     /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(6168..6301,1)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6205..6224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6259..6278
                     /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 gctctgttac ttaaggcttt tatatcaacg atgttacctc gttttagttc taagtttcgt
     3901 gataagtttt gacttacatg caacatgtag cttttagaaa agatttttgt ttttcatgtt
     3961 tttttttatt acaatttcat ttttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4021 aaaaaaaaaa aaaaaagtac cgcggccgct cgagtctaga gggcccgcgg ttcgaaggta
     4081 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtcat catcaccatc
     4141 accattgagt ttatctgact aaatcttagt ttgtattgtc atgttttaat acaatatgtt
     4201 atgtttaaat atgtttttaa taaattttat aaaataattt caacttttat tgtaacaaca
     4261 ttgtccattt acacactcct ttcaagcgcg tgggatcgat gctcactcaa aggcggtaat
     4321 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4381 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4441 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4501 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4561 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4621 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4681 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4741 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4801 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4861 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4921 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4981 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5041 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgatgataa acaatgtatg
     5101 gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt gccaacaagc
     5161 acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa aaacacgctt
     5221 ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt tacataaata
     5281 gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt ttgcagtgca
     5341 aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc tgtcacgtac
     5401 gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc agcttcctgt
     5461 gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc catatcagcc
     5521 gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat cgcgcctata
     5581 aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga attaattcgg
     5641 gatctgccgg gctgcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     5701 ataatacgac tcactatagg agggccacca tggccaagtt gaccagtgcc gttccggtgc
     5761 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     5821 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     5881 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     5941 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     6001 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     6061 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgaccgacg ccgaccaaca
     6121 ccgccggtcc gacgcggccc gacgggtccg aggggggtcg acctcgaaac ttgtttattg
     6181 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6241 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     6301 t
//
