LOCUS       sequence-367527-        3460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piwi sgRNA plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3460)
  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 3460)
  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: piwi_sgRNA
FEATURES             Location/Qualifiers
     source          1..3460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..123
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     115..132
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     116..132
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     139..158
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        139..157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     182..198
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     183..199
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        211..610
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        631..706
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(735..751)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(735..751)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(788..806)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(827..843)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(827..843)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(840..862)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    851..867
                     /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(875..881,882..899,900..905))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    920..941
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1058..1075)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1229..1817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1309..1328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1988..2779,2780..2848))
                     /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     2611..2630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2849..2953)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2979..3434)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3116..3137)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3328..3347
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
       61 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
      121 acgacggcca gtgaattgta atacgactca ctatagggcg aattgggtac cgggcccccc
      181 ctcgaggtcg acggtatcga taagcttgat gttcgacttg cagcctgaaa tacggcacga
      241 gtaggaaaag ccgagtcaaa tgccgaatgc agagtctcat tacagcacaa tcaactcaag
      301 aaaaactcga cactttttta ccatttgcac ttaaatcctt ttttattcgt tatgtatact
      361 ttttttggtc cctaaccaaa acaaaaccaa actctcttag tcgtgcctct atatttaaaa
      421 ctatcaattt attatagtca ataaatcgaa ctgtgttttc aacaaacgaa caataggaca
      481 ctttgattct aaaggaaatt ttgaaaatct taagcagagg gttcttaaga ccatttgcca
      541 attcttataa ttctcaactg ctctttcctg atgttgatca tttatatagg tatgttttcc
      601 tcaatacttc gtaacaatgg ctgatgatca gttttagagc tagaaatagc aagttaaaat
      661 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttatcgaatt
      721 cctgcagccc gggggatcca ctagttctag agcggccgcc accgcggtgg agctccagct
      781 tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
      841 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
      901 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
      961 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1021 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1081 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1141 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1201 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1261 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1321 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1381 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1441 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1501 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1561 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1621 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     1681 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     1741 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     1801 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     1861 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     1921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2281 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     2581 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     2641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     2701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     2761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     2821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     2881 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2941 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     3001 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3061 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3121 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3181 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3241 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3301 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3361 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3421 gctacagggc gcgtcccatt cgccattcag gctgcgcaac
//
