LOCUS       sequence-367525-        5467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously tag piwi gene in Drosophila 
            melanogaster ovarian somatic sheet (OSS) cells. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5467)
  AUTHORS   Meng Q, Stoyko D, Andrews CM, Konstantinidou P, Genzor P, O T, 
            Elchert AR, Benner L, Sobti S, Katz EY, Haase AD
  TITLE     Functional editing of endogenous genes through rapid selection of 
            cell pools (Rapid generation of endogenously tagged genes in 
            Drosophila ovarian somatic sheath cells).
  JOURNAL   Nucleic Acids Res. 2022 May 27. pii: 6594084. doi: 
            10.1093/nar/gkac448.
  PUBMED    35639929
REFERENCE   2  (bases 1 to 5467)
  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"; journalName: "Nucleic 
            Acids Res. 2022 May 27. pii: 6594084. doi: 10.1093/nar/gkac448."
COMMENT     Sequence Label: Donor_N_FH_Piwi
FEATURES             Location/Qualifiers
     source          1..5467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..63
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    180..201
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(216..221,222..239,240..246)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    254..270
                     /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)."
     primer_bind     259..281
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     278..294
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     278..294
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1080..1214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1104..1123)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1158..1177
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1215..1814)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1298..1318)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1795..1814
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             2169..2234
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2271..2360
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(3145..3162)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3145..3161)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3154..3176)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3370..3389)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3489..3511
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3549..3567)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3635..3739
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3740..3808,3809..4600)
                     /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     complement(3958..3977)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4771..5359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5260..5279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
       61 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      121 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      181 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      241 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      301 tacgccaagc ttgcatgctc aaaatattca atttccaagt catcccgtac aatttcaata
      361 attctgcgaa ggttttcctt agtgtatcgg tactttagtc gaaatttatg ctcatttaga
      421 tcggtcaatg gattaaatct tgggttcctt ttctttatat gcgttgcccg cgctcgagaa
      481 ttgtgtttga tattcagaaa acattgaaag tctttgaaat aatcaaagtc ttttagagac
      541 ctttgaaatc tttgtatatc ttccattttc acttgtttac ttgcatagca gggtggctgt
      601 tttgtatata ttagaaaata cctcatacgt acatgcggta tttaaggctc ttgcaacgat
      661 tgaaatttta tgatgaaact ttacacttta aaaataatct ttttaaatgt tgttaattaa
      721 aaaaatgtaa gtaagtaaat taacactttt atttttttta agcacgttat gtacttttgg
      781 agttaaataa tttttttttt atgtatttct atacttttaa agaaacagaa cagaaagaaa
      841 tcaaagctcg aagtaccaaa aaataaatac aagggaaata gaacctattg cgaagatttg
      901 caaaagcgat tgttttcagt tgtgaagact aatatctaca cattccaaaa gcttaacgat
      961 ctttgaaatc tggccatttg gtcacactga gtccaaagcg tcgttttcaa agtactcttt
     1021 cagtttccat tgtgaagttt taagtgatcg cgagtgccaa aaagtaacac caggtaagtg
     1081 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     1141 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     1201 gcaataaaca agttctaggc accgggcttg cgggtcatgc accaggtgcg cggtccttcg
     1261 ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg
     1321 ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc cactccgggg
     1381 agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagacgcc gacggtggcc
     1441 aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat ctgttgctgc
     1501 gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc gaacaccgcc
     1561 cccgcttcga cgctctccgg cgtggtccag accgccaccg cggcgccgtc gtccgcgacc
     1621 cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc
     1681 cgctcgatgt ggcggtccgg gtcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg
     1741 gcggcgaggg tgcgtacggc ccgggggacg tcgtcgcggg tggcgaggcg caccgtgggc
     1801 ttgtactcgg tcattttgat tgtctctgga ttagacgact gctggctgat ggagcggctt
     1861 tgtgtcggga ggagtatcca cacagcacaa gaactcaaac ggtagtgata tgaaaactgg
     1921 tcccgcagcc cgttttataa agcccgctta gcgctaaaac cttatcagcc gcaatgctct
     1981 ccgctctcaa gtcgcgttca aaacttttac catatatggg tatgaattgt tcgccgattg
     2041 ggttttcatt tacggagaat ttcctccgca actgggtgtt cgtgcttgtg tgtgtcttgt
     2101 atatcgccat gaagagtgta cctaatttta atcaaccgat ttctctctct tccgcaggaa
     2161 tcaaaatgga ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg
     2221 atgacgatga caagggtgga tctggaggtt ccggcggctc agggggtagt tacccatacg
     2281 atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga tcctatccat
     2341 atgacgttcc agattacgct ggtggatctg gaggttccgg cggctcaggg ggtagtgctg
     2401 atgatcaggg acgtggacgc aggcgtccac ttaacgaaga tgattcctct acttcccgag
     2461 gtagtggtga tgggccggta tgtaaactct tattcaagct aggaaaaaaa atcatagcgg
     2521 taaagcaaat tccttataat tccgttatgt cgcaaacaca aacacagcca tgtacataga
     2581 ttgtaaatgc gcgcatttct tcggaattcg acaccaaaag gaagcactaa aactacaagt
     2641 caaataaaca aacaaaaatc tgctatgaat ttgagcttga ctgcataaaa tcttcttgaa
     2701 tttctgttga ccaagcactt ttttcaaagg gaattcatta aacatatttc caaaaacgct
     2761 tctttcaatt tgaatttcca aggtaatgta aagcattatc tgtttcgttt catttatttt
     2821 tttttaataa aaactgctaa aataaccatc acctgacagg aaggtggctg tggcaagccg
     2881 aatcttattt acccttgcag tctatgtctt tggaatctgc atgcccgacg ttgatatgga
     2941 cggacatgac caaatggacc cggctagtga tactcatcaa aaatcatggt cggaaacgct
     3001 tccttctatc tgtcacatac atttgattcc tagaaactct acgagtaacg ggtatttctc
     3061 cgatattcat cccaattcta tacaactttg agatggcttt cagcattgat atactctgaa
     3121 attgcaggca aagcatacga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3181 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3241 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3301 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     3361 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3421 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3481 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3541 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3601 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3661 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3721 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3781 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3841 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3901 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3961 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4021 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4081 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4141 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4201 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4261 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4321 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4381 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4441 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4501 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4561 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4621 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4681 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4741 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4801 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4861 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4921 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4981 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5041 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5101 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5161 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5221 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5281 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5341 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5401 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5461 taccgcc
//
