LOCUS       sequence-362364-        5454 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 5454)
  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 5454)
  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..5454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1005..1139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1029..1048)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1083..1102
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1140..1739)
                     /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(1223..1243)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1720..1739
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             2094..2159
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2196..2285
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(3058..3075)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3058..3074)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3067..3089)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3283..3302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3402..3424
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3462..3480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3548..3652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3653..3721,3722..4513)
                     /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(3871..3890)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4684..5272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5173..5192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5426..5443
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgctcaaaa tattcaattt ccaagtcatc ccgtacaatt tcaataattc tgcgaaggtt
      301 ttccttagtg tatcggtact ttagtcgaaa tttatgctca tttagatcgg tcaatggatt
      361 aaatcttggg ttccttttct ttatatgcgt tgcccgcgct cgagaattgt gtttgatatt
      421 cagaaaacat tgaaagtctt tgaaataatc aaagtctttt agagaccttt gaaatctttg
      481 tatatcttcc attttcactt gtttacttgc atagcagggt ggctgttttg tatatattag
      541 aaaatacctc atacgtacat gcggtattta aggctcttgc aacgattgaa attttatgat
      601 gaaactttac actttaaaaa taatcttttt aaatgttgtt aattaaaaaa atgtaagtaa
      661 gtaaattaat acttttattt tttttaagca cgttatgtac ttttggagtt aaataatttt
      721 tttttatgta tttctatact tttaaaaaaa cagaacagaa agaaatcaaa gctcgaagta
      781 ccaaaaaata aatactaggg aaatagaacc tattgcgaag atttgcaaaa gcgattgttt
      841 tcagttgtga agactaatat ctacacattc caaaagctta acgatctttg aaatctggcc
      901 atttggtcac actgagtcca aagcgtcgtt ttcaaagtac tctttcagtt tccattgtga
      961 agttttaagt gatcgcgagt gccaaaaagt aacaccaggt aagtgatcca gacatgataa
     1021 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1081 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttc
     1141 taggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     1201 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc
     1261 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     1321 cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc
     1381 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     1441 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     1501 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     1561 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     1621 tccgggtcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     1681 acggcccggg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatt
     1741 ttgattgtct ctggattaga cgactgctgg ctgatggagc ggctttgtgt cgggaggagt
     1801 atccacacag cacaagaact caaacggtag tgatatgaaa actggtcccg cagcccgttt
     1861 tataaagccc gcttagcgct aaaaccttat cagccgcaat gctctccgct ctcaagtcgc
     1921 gttcaaaact tttaccatat atgggtatga attgttcgcc gattgggttt tcatttacgg
     1981 agaatttcct ccgcaactgg gtgttcgtgc ttgtgtgtgt cttgtatatc gccatgaaga
     2041 gtgtacctaa ttttaatcaa ccgatttctc tctcttccgc aggaatcaaa atggactaca
     2101 aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagg
     2161 gtggatctgg aggttccggc ggctcagggg gtagttaccc atacgatgtt cctgactatg
     2221 cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac gttccagatt
     2281 acgctggtgg atctggaggt tccggcggct cagggggtag tgctgatgat cagggacgtg
     2341 gacgcaggcg tccacttaac gaagatgatt cctctacttc ccgaggtagt ggtgatgggc
     2401 cggtatgtaa actcttattc aagctaggaa aaaaaatcat agcggtaaag caaattcctt
     2461 ataattccgt tatgtcgcaa acacaaacac agccatgtac atagattgta aatgcgcgca
     2521 tttcttcgga attcgacacc aaaaggaagc actaaaacta acaaacaaaa atctgctatg
     2581 aatttgagct tgactgcata aaatcttctt gaatttctgt tgaccaagca cttttttcaa
     2641 agggaattca ttaaacatat ttccaaaaac gcttctttca atttgaattt ccaaggaaat
     2701 gtaaagcatt atctgtttcg tttcatttat ttttttttaa taaaaactgc taaaataacc
     2761 atcacctgac aggaaggtgg ctgtggcaag ccgaatctta tttacccttg cagtctatgt
     2821 ctttggaatc tgcatgcccg acgttgatat ggacggacat gaccaaatgg acccggctag
     2881 tgatactcat caaaaatcat ggtcggaaac gcttccttct atctgtcaca tacatttgat
     2941 tcctagaaac tctacgagta acgggtattt ctccgatatt catcccaatt ctatacaact
     3001 ttgagatggc tttcagcatt gatatactct gaaattgcag gcaaagcata cgaattcact
     3061 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3121 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3181 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     3241 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     3301 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3361 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3421 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     3481 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3541 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3601 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3661 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3721 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3781 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3841 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3901 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3961 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4021 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4081 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4141 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4201 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4261 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4321 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4381 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4441 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4501 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4561 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4621 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4681 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4741 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4801 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4861 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4921 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4981 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5041 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5101 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5161 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5221 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5281 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5341 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5401 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaga
//
