LOCUS       sequence-259371-        8151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express arrays of gRNA targeting Wingless under dU6-3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8151)
  AUTHORS   Buchman AB, Brogan DJ, Sun R, Yang T, Hsu PD, Akbari OS
  TITLE     Programmable RNA Targeting Using CasRx in Flies.
  JOURNAL   CRISPR J. 2020 Jun;3(3):164-176. doi: 10.1089/crispr.2020.0018.
  PUBMED    32584145
REFERENCE   2  (bases 1 to 8151)
  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"; doi: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-06"; 
            volume: "3"; issue: "3"; pages: "164-176"
COMMENT     Sequence Label: OA-1050V (wingless)
FEATURES             Location/Qualifiers
     source          1..8151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1145..1216,1618..1891,1966..2620,2682..2997,3201..3332,
                     3403..4017)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4792..4825
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4884..5304
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5313..5348
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        5379..5414
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        5445..5480
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        5511..5546
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        5577..5612
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     protein_bind    5675..5744
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(5963..5981)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6002..6018)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6002..6018)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6015..6037)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6026..6042
                     /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(6050..6056,6057..6074,6075..6080))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6095..6116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6233..6250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6404..6992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6484..6503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7163..7954,7955..8023))
                     /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     7786..7805
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8024..8128)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt ttaaatttac
      781 ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact caaaatgaca
      841 ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat cgccgtctct
      901 ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta gcgtcaatgt
      961 ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg tttacgccat
     1021 caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg tttgtttttt
     1081 gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt gaaaaatccc
     1141 ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac acccatctgc
     1201 cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac ttgatctata
     1261 ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc acaaaaatcc
     1321 caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt agctgttctc
     1381 gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg cattcatttt
     1441 cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg atcctagatg
     1501 cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt actttttgtt
     1561 cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc attgcagggt
     1621 gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc caaaaactac
     1681 ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg ccaactagcc
     1741 gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa tcagccgggc
     1801 tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga gcgacacata
     1861 ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt cggctgatct
     1921 gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc ggcgtggcct
     1981 atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg accctgctga
     2041 atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg atgcgactgc
     2101 tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat gtccagcagg
     2161 atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccaa gccatggtgc
     2221 ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag gtgatccagg
     2281 agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg gtgaaaggtc
     2341 tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggctctaacc gatccgccgc
     2401 ttctgatctg cgatgagccc acctccggac tggactcctt taccgcccac agcgtcgtcc
     2461 aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt catcagccgt
     2521 cttccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc agggtagctt
     2581 tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga tgtgtttatt
     2641 aagggtatct agtattacat aacatctcaa ctcctatcca gcgtgggtgc ccagtgtcct
     2701 accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc cggacgggag
     2761 atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgccattag caaagtagcc
     2821 cgggatatgg agcagttgtt ggccaccaaa aatctggaga agccactgga gcagccggag
     2881 aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct gtggcgatcc
     2941 tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca gacaacggtg
     3001 agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca aattcgctag
     3061 attttagata gaattgcctg attccacacc cttcttagtt tttttcaatg agatgtatag
     3121 tttatagttt tgcagaagat aaataaattt catttaactc gcgaatatta atgagatgcg
     3181 agtaacattt taatttgcag atggttgcca tcttgattgg cctcatcttt ttgggccaac
     3241 aactcacgca agtgggtgtg atgaatatca acggagccat cttcctcttc ctgaccaaca
     3301 tgacctttca aaacgtcttt gccacgataa atgtaagtca tgtttagaat acatttgcat
     3361 ttcaataatt tactaacttt ctaatgaatc gattcgattt aggtgttcac ctcagagctg
     3421 ccagttttta tgagggaggc ccgaagtcga ctttatcgct gtgacacata ctttctgggc
     3481 aaaacgattg ccgaattgcc gctttttctc acagtgccac tggtcttcac ggcgattgcc
     3541 tatccgatga tcggactgcg ggccggagtg ctgcacttct tcaactgcct ggcgctggtc
     3601 actctggtgg ccaatgtgtc aacgtccttc ggatatctaa tatcctgcgc cagctcctcg
     3661 acctcgatgg cgctgtctgt gggtccgccg gttatcatac cattcctgct ctttggcggc
     3721 ttcttcttga actcgggctc ggtgccagta tacctcaaat ggttgtcgta cctctcatgg
     3781 ttccgttacg ccaacgaggg tctgctgatt aaccaatggg cggacgtgga gccgggcgaa
     3841 attagctgca catcgtcgaa caccacgtgc cccagttcgg gcaaggtcat cctggagacg
     3901 cttaacttct ccgccgccga tctgccgctg gactacgtgg gtctggccat tctcatcgtg
     3961 agcttccggg tgctcgcata tctggctcta agacttcggg cccgacgcaa ggagtagccg
     4021 acatatatcc gaaataactg cttgtttttt ttttttacca ttattaccat cgtgtttact
     4081 gtttattgcc ccctcaaaaa gctaatgtaa ttatatttgt gccaataaaa acaagatatg
     4141 acctatagaa tacaagtatt tccccttcga acatccccac aagtagactt tggatttgtc
     4201 ttctaaccaa aagacttaca cacctgcata ccttacatca aaaactcgtt tatcgctaca
     4261 taaaacaccg ggatatattt tttatataca tacttttcaa atcgcgcgcc ctcttcataa
     4321 ttcacctcca ccacaccacg tttcgtagtt gctctttcgc tgtctcccac ccgctctccg
     4381 caacacattc accttttgtt cgacgacctt ggagcgactg tcgttagttc cgcgcgattc
     4441 ggttcgctca aatggttccg agtggttcat ttcgtctcaa tagaaattag taataaatat
     4501 ttgtatgtac aatttatttg ctccaatata tttgtatata tttccctcac agctatattt
     4561 attctaattt aatattatga ctttttaagg taattttttg tgacctgttc ggagtgatta
     4621 gcgttacaat ttgaactgaa agtgacatcc agtgtttgtt ccttgtgtag atgcatctca
     4681 aaaaaatggt gggcataata gtgttgttta tatatatcaa aaataacaac tataataata
     4741 agaatacatt taatttagaa aatgcttgga tttcactgga actaggctag cataacttcg
     4801 tataatgtat gctatacgaa gttatgctag cggatccggg aattgggaat tgggcaatat
     4861 ttaaatggcg gcgcgccgaa ttcttttttg ctcacctgtg attgctccta ctcaaataca
     4921 aaaacatcaa attttctgtc aataaagcat atttatttat atttatttta caggaaagaa
     4981 ttccttttaa agtgtatttt aacctataat gaaaaacgat taaaaaaaat acataaaata
     5041 attcgaaaat ttttgaatag cccaggttga taaaaattca tttcatacgt tttataactt
     5101 atgcccctaa gtattttttg accatagtgt ttcaattcta cattaatttt acagagtaga
     5161 atgaaacgcc acctactcag ccaagaggcg aaaaggttag ctcgccaagc agagagggcg
     5221 ccagtgctca ctacttttta taattctcaa cttctttttc cagactcagt tcgtatatat
     5281 agacctattt tcaatttaac gtcggctgca ggcaagtaaa cccctaccaa ctggtcgggg
     5341 tttgaaacgt ggatcgctgg gtccatgtac atgatgggca agtaaacccc taccaactgg
     5401 tcggggtttg aaacacactg cccgcctggt ggttcgcttg tggacaagta aacccctacc
     5461 aactggtcgg ggtttgaaac tattatgctt gcgtccctga cgacgtccat caagtaaacc
     5521 cctaccaact ggtcggggtt tgaaactcga cgcccagcga ggtctcattg cactggcaag
     5581 taaaccccta ccaactggtc ggggtttgaa actttttttg cggccgcaac ggtacctcta
     5641 gagatccact agtgtcgacg atgtaggtca cggtctcgaa gccgcggtgc gggtgccagg
     5701 gcgtgccctt gggctccccg ggcgcgtact ccacctcacc catctggtcc atcatgatga
     5761 acgggtcgag gtggcggtag ttgatcccgg cgaacgcgcg gcgcaccggg aagccctcgc
     5821 cctcgaaacc gctgggcgcg gtggtcacgg tgagcacggg acgtgcgacg gcgtcggcgg
     5881 gtgcggatac gcggggcagc gtcagcgggt tctcgacggt cacggcgggc atgtcgacac
     5941 tagttctagc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
     6001 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6061 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6121 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6181 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6241 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6301 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6361 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6421 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6481 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6541 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6601 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6661 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6721 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6781 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6841 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6901 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6961 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7021 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7081 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7141 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7201 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7261 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7321 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7381 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7441 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7501 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7561 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7621 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7681 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7741 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7801 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7861 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7921 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7981 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8041 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8101 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
