LOCUS       sequence-214681-        9229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA cloning vector for expressing 1-3 gRNAs in Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9229)
  AUTHORS   Poe AR, Wang B, Sapar ML, Ji H, Li K, Onabajo T, Fazliyeva R, Gibbs 
            M, Qiu Y, Hu Y, Han C
  TITLE     Robust CRISPR/Cas9-Mediated Tissue Specific Mutagenesis Reveals Gene
            Redundancy and Perdurance in Drosophila.
  JOURNAL   Genetics. 2018 Nov 30. pii: genetics.118.301736. doi: 
            10.1534/genetics.118.301736.
  PUBMED    30504366
REFERENCE   2  (bases 1 to 9229)
  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: "Genetics. 
            2018 Nov 30. pii: genetics.118.301736. doi: 
            10.1534/genetics.118.301736."
COMMENT     Sequence Label: pAC-U61-SapI
FEATURES             Location/Qualifiers
     source          1..9229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    join(405..606,607..637)
                     /label=P element 3' end
                     /note="P element 3' end"
     protein_bind    1181..1250
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(1460..1479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1579..1601
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1639..1657)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1725..1829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1830..1898,1899..2690)
                     /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(2048..2067)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2861..3449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3350..3369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(3684..3714,3715..4269)
                     /label=P element 5' end
     CDS             complement(join(5048..5662,5733..5864,6068..6383,6445..7099,
                     7174..7447,7849..7920))
                     /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"
     primer_bind     8372..8390
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     promoter        8433..9229
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
ORIGIN
        1 ggaagagcga ggctcttctg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgcctacctg gagcctgaga
      121 gttgttcaat aaaataaaaa tgtttcgttt ttttgctttc gccagtattt attatttttc
      181 atcaatatgt attcaatttg gtatgtattt agtaattgta atatatagac aatggttttc
      241 cgttgacgta catacatctg acgtgtgttt atttagacat aatagttatg ttttcacatc
      301 tttttaatgt tcgcttaatg cgtatgcatt ctagagtcga cctcgaacgt taacgttaac
      361 gtaacgttaa ctcgaggcct cgaggtcgac ctgcagccaa gctttgcgta ctcgcaaatt
      421 attaaaaata aaactttaaa aataatttcg tctaattaat attatgagtt aattcaaacc
      481 ccacggacat gctaagggtt aatcaacaat catatcgctg tctcactcag actcaatacg
      541 acactcagaa tactattcct ttcactcgca cttattgcaa gcatacgtta agtggatgtc
      601 tcttgccgac gggaccacct tatgttattt catcatggtc tggccattct catcgtgagc
      661 ttccgggtgc tcgcatatct ggctctaaga cttcgggccc gacgcaagga gtagccgaca
      721 tatatccgaa ataactgctt gttttttttt tttaccatta ttaccatcgt gtttactgtt
      781 tattgccccc tcaaaaagct aatgtaatta tatttgtgcc aataaaaaca agatatgacc
      841 tatagaatac aagtatttcc ccttcgaaca tccccacaag tagactttgg atttgtcttc
      901 taaccaaaag acttacacac ctgcatacct tacatcaaaa actcgtttat cgctacataa
      961 aacaccggga tatatttttt atatacatac ttttcaaatc gcgcgccctc ttcataattc
     1021 acctccacca caccacgttt cgtagttgct ctttcgctgt ctcccacccg ctctccgcaa
     1081 cacattcacc ttttgttcga cgaccttgga gcgactgtcg ttagttccgc gcgattcggt
     1141 gcggtatttc acaccgcata tcgacgatgt aggtcacggt ctcgaagccg cggtgcgggt
     1201 gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc tggtccatca
     1261 tgatgaacgg gtcgaggtgg cggtagttga tcccggcgaa cgcgcggcgc accgggaagc
     1321 cctcgccctc gaaaccgctg ggcgcggtgg tcacggtgag cacgggacgt gcgacggcgt
     1381 cggcgggtgc ggatacgcgg ggcagcgtca gcgggttctc gacggtcacg gcgggcatgt
     1441 cgatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     1501 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1561 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     1621 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     1681 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1741 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1801 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1861 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1921 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1981 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2041 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2101 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2161 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2221 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2281 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2341 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2401 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2461 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2521 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2581 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2641 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2701 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2761 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2821 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2881 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2941 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3001 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3061 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3121 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3181 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3241 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3301 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3361 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3421 gctcgtcagg ggggcggagc ctatggaaaa acgccttctt cttgaactcg ggctcggtgc
     3481 cagtatacct caaatggttg tcgtacctct catggttccg ttacgccaac gagggtctgc
     3541 tgattaacca atgggcggac gtggagccgg gcgaaattag ctgcacatcg tcgaacacca
     3601 cgtgccccag ttcgggcaag gtcatcctgg agacgcttaa cttctccgcc gccgatctgc
     3661 cgctggacta cgtgggtctg gcccatgatg aaataacata aggtggtccc gtcgatagcc
     3721 gaagcttacc gaagtataca cttaaattca gtgcacgttt gcttgttgag aggaaaggtt
     3781 gtgtgcggac gaattttttt ttgaaaacat taacccttac gtggaataaa aaaaaatgaa
     3841 atattgcaaa ttttgctgca aagctgtgac tggagtaaaa ttaattcacg tgccgaagtg
     3901 tgctattaag agaaaattgt gggagcagag ccttgggtgc agccttggtg aaaactccca
     3961 aatttgtgat acccacttta atgattcgca gtggaaggct gcacctgcaa aaggtcagac
     4021 atttaaaagg aggcgactca acgcagatgc cgtacctagt aaagtgatag agcctgaacc
     4081 agaaaagata aaagaaggct ataccagtgg gagtacacaa acagagtaag tttgaatagt
     4141 aaaaaaaatc atttatgtaa acaataacgt gactgtgcgt taggtcctgt tcattgttta
     4201 atgaaaataa gagcttgagg gaaaaaattc gtactttgga gtacgaaatg cgtcgtttag
     4261 agcagcagcc gaattaattc tagttccagt gaaatccaag cattttctaa attaaatgta
     4321 ttcttattat tatagttgtt atttttgata tatataaaca acactattat gcccaccatt
     4381 tttttgagat gcatctacac aaggaacaaa cactggatgt cactttcagt tcaaattgta
     4441 acgctaatca ctccgaacag gtcacaaaaa attaccttaa aaagtcataa tattaaatta
     4501 gaataaatat agctgtgagg gaaatatata caaatatatt ggagcaaata aattgtacat
     4561 acaaatattt attactaatt tctattgaga cgaaatgaac cactcggaac catttgagcg
     4621 aaccgaatcg cgcggaacta acgacagtcg ctccaaggtc gtcgaacaaa aggtgaatgt
     4681 gttgcggaga gcgggtggga gacagcgaaa gagcaactac gaaacgtggt gtggtggagg
     4741 tgaattatga agagggcgcg cgatttgaaa agtatgtata taaaaaatat atcccggtgt
     4801 tttatgtagc gataaacgag tttttgatgt aaggtatgca ggtgtgtaag tcttttggtt
     4861 agaagacaaa tccaaagtct acttgtgggg atgttcgaag gggaaatact tgtattctat
     4921 aggtcatatc ttgtttttat tggcacaaat ataattacat tagctttttg agggggcaat
     4981 aaacagtaaa cacgatggta ataatggtaa aaaaaaaaaa caagcagtta tttcggatat
     5041 atgtcggcta ctccttgcgt cgggcccgaa gtcttagagc cagatatgcg agcacccgga
     5101 agctcacgat gagaatggcc agacccacgt agtccagcgg cagatcggcg gcggagaagt
     5161 taagcgtctc caggatgacc ttgcccgaac tggggcacgt ggtgttcgac gatgtgcagc
     5221 taatttcgcc cggctccacg tccgcccatt ggttaatcag cagaccctcg ttggcgtaac
     5281 ggaaccatga gaggtacgac aaccatttga ggtatactgg caccgagccc gagttcaaga
     5341 agaagccgcc aaagagcagg aatggtatga taaccggcgg acccacagac agcgccatcg
     5401 aggtcgagga gctggcgcag gatattagat atccgaagga cgttgacaca ttggccacca
     5461 gagtgaccag cgccaggcag ttgaagaagt gcagcactcc ggcccgcagt ccgatcatcg
     5521 gataggcaat cgccgtgaag accagtggca ctgtgagaaa aagcggcaat tcggcaatcg
     5581 ttttgcccag aaagtatgtg tcacagcgat aaagtcgact tcgggcctcc ctcataaaaa
     5641 ctggcagctc tgaggtgaac acctaaatcg aatcgattca ttagaaagtt agtaaattat
     5701 tgaaatgcaa atgtattcta aacatgactt acatttatcg tggcaaagac gttttgaaag
     5761 gtcatgttgg tcaggaagag gaagatggct ccgttgatat tcatcacacc cacttgcgtg
     5821 agttgttggc ccaaaaagat gaggccaatc aagatggcaa ccatctgcaa attaaaatgt
     5881 tactcgcatc tcattaatat tcgcgagtta aatgaaattt atttatcttc tgcaaaacta
     5941 taaactatac atctcattga aaaaaactaa gaagggtgtg gaatcaggca attctatcta
     6001 aaatctagcg aatttgtttc caagaattgt aagcgttata tcatttgttt ccactggaac
     6061 cactcaccgt tgtctgaata agtcgcactt ttacgaggag tggttccttg agcaccgaca
     6121 gccaggatcg ccacaggacc gcccggaact gcatgaacca ggtggccttg taggtgtacc
     6181 cattctccgg ctgctccagt ggcttctcca gatttttggt ggccaacaac tgctccatat
     6241 cccgggctac tttgctaatg gcaaaattgt cgcatatctt ggcgatccga tcacgggact
     6301 cgatctcccg tccgggcaca acggccaaca cctgtacgta aaagtccgcc ggattgtagt
     6361 tggtaggaca ctgggcaccc acgctggata ggagttgaga tgttatgtaa tactagatac
     6421 ccttaataaa cacatcgaac tcactaggaa aagaagtcga cggcttcgct gggagtgccc
     6481 aagaaagcta ccctgccctc ggccatcaga aggatcttgt caaagagctc aaacagctcg
     6541 gaagacggct gatgaatggt caggatgacg gtcttgccct tctgcgacag cttcttcagc
     6601 acctggacga cgctgtgggc ggtaaaggag tccagtccgg aggtgggctc atcgcagatc
     6661 agaagcggcg gatcggttag agcctcggag gcgaatgcca gacgcttcct ttctccgccg
     6721 gacagacctt tcaccctgcc gggcacaccg atgatcgtgt gctgacattt gctgagcgaa
     6781 agctcctgga tcacctgatc cacgcgggcc actcgctgcc gataggtcag atgtcgtggc
     6841 atccgcacca tggcttggaa aatcaggtgt tccctggccg ttagggagcc gataaagagg
     6901 tcatcctgct ggacataggc gcacctggcc tgcatctcct tggcgtccac aggttggcca
     6961 ttgagcagtc gcatcccgga tggcgatact tggatgccct gcggcgatcg aaaggcaagg
     7021 gcattcagca gggtcgtctt tccggcaccg gaactgccca tcacggccaa aagttcgccc
     7081 ggataggcca cgccgcaaac tgagtttcaa attggtaatt ggacccttta ttaagatttc
     7141 acacagatca gccgactgcg aatagaaact caccgttctt gagcaaatgt ttcctgggcg
     7201 ccggtatgtg tcgctcgttg cagaatagtc cgcgtgtccg gttgaccagc tgccgccatc
     7261 cggagcccgg ctgattgacc gccccaaaga tgtccatatt gtgccaggca taggtgaggt
     7321 tctcggctag ttggccgctc cctgaaccgg agtcctccgg cggactgggt ggccggagcg
     7381 tgccgtagtt tttggcctgc ccgaagccct ggttaatgca gctctgcgaa gccgctccgc
     7441 tgtcaccctg caatgatagg ggatctcaaa tatcaactac aagcgttatg ctcatctaac
     7501 cccgaacaaa aagtaccccg aagtatccta cgaagtaggt ttatactttt atttattttt
     7561 tgtgcatcta ggatcagctt aaaatatctg gttgttatat tttttgtaaa aaagaatata
     7621 gtcgaaaatg aatgccttta gatgtcttga tcatgatatg atctcaaaaa ttgtcttata
     7681 tagcgagaac agctaccaga ataatctgtt tcgtgtcact atttgtttgt gcaattgcgg
     7741 tttgggattt ttgtgggtcg cagttctcac gccgcagaca atttgatgtt gcaatcgcag
     7801 ttcctataga tcaagtgaac ttaagatgta tgcacatgta ctactcacat tgttcagatg
     7861 ctcggcagat gggtgtttgc tgcctccgcg aattaatagc tcctgatcct cttggcccat
     7921 tgccgggatt tttcacactt tcccctgctt acccacccaa aaccaatcac caccccaatc
     7981 actcaaaaaa caaacaaaaa taagaagcga gaggagtttt ggcacagcac tttgtgttta
     8041 attgatggcg taaaccgctt ggagcttcgt cacgaaaccg ctgacaaaat gcaactgaag
     8101 gcggacattg acgctacgta acgctacaaa cggtggcgaa agagatagcg gacgcagcgg
     8161 cgaaagagac ggcgatattt ctgtggacag agaaggaggc aaacagcgct gactttgagt
     8221 ggaatgtcat tttgagtgag aggtaatcga aagaacctgg tacatcaaat acccttggat
     8281 cgaagtaaat ttaaaactga tcagataagt tcaatgatat ccagtgcagt aaaaaaaaaa
     8341 aatgtttttt ttatctactt tccgcaaaaa tgggttttat taacttacat acatactaga
     8401 attaggccac tagtggatct ggatctgaat tcattttcaa cgtcctcgat agtatagtgg
     8461 ttagtatccc cgcctgtcac gcgggagacc ggggttcaat tccccgtcgg ggagaatctg
     8521 tgattctttt tttttttctt ttactttgtt atataaacaa tttttgtttt aattgaatct
     8581 aatttgccat tgcttttagg aatctcaggc atccagcaag cgtttgtccg ccgaatcgcc
     8641 catcagtgaa gaagatcctg tggcggctac gaaaatctcc ccggccatgt cggcctccac
     8701 ctccagcgaa aaacccatca gcgagctggc cacctctgtg ctgacccacc gctttccaga
     8761 ctccacctcc tcacccggcg aacatggcct tggacgaatg cagttgtcga tccgctacag
     8821 cgcccagcgt caaaaactag acgtgaccat acacaaaatc cagaagatac cacttcgcga
     8881 tcccagcaat atccccgatc cgtatgttaa gctgtatctg ttgcctggac gcaccaagga
     8941 gtcgaaacgc aagacgagcg tgatcaagga caactgcaac cccgtctacg atgcatcctt
     9001 tgagtacctg atttccattg ccgaactcag gcagacggaa ctggaggtga cggtgtgcac
     9061 ccaaaaggga ttcctatccg gcggtagtcc catcattggc atggtaggta cccgaaagca
     9121 accccttagt tacagacaca gcgcgtacgt ccttcgcatc cttatgattc ccaagtacat
     9181 attctgcaag agtacagtat atataggaaa gatatccggg tgaacttcg
//
