LOCUS       sequence-237079-        8967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA cloning vector for expressing 2-6 gRNAs based on tgFE design in
            Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8967)
  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 8967)
  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-U63-tgRNA-Rev
FEATURES             Location/Qualifiers
     source          1..8967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     141..163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(201..219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        287..391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(392..460,461..1252)
                     /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(610..629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1423..2011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1912..1931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(2246..2276,2277..2831)
                     /label=P element 5' end
     CDS             complement(join(3610..4224,4295..4426,4630..4945,5007..5661,
                     5736..6009,6411..6482))
                     /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     6934..6952
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     promoter        complement(7420..7840)
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_feature    join(7934..8135,8136..8166)
                     /label=P element 3' end
                     /note="P element 3' end"
     protein_bind    8710..8779
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gtcgatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
       61 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      181 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
      241 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      361 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      421 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      481 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      541 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      601 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
      661 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
      721 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
      781 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
      841 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
      901 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
      961 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1021 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1081 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1141 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1201 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1261 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1321 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1381 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1441 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1501 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1561 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1621 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1681 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1741 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1801 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1861 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1921 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1981 atgctcgtca ggggggcgga gcctatggaa aaacgccttc ttcttgaact cgggctcggt
     2041 gccagtatac ctcaaatggt tgtcgtacct ctcatggttc cgttacgcca acgagggtct
     2101 gctgattaac caatgggcgg acgtggagcc gggcgaaatt agctgcacat cgtcgaacac
     2161 cacgtgcccc agttcgggca aggtcatcct ggagacgctt aacttctccg ccgccgatct
     2221 gccgctggac tacgtgggtc tggcccatga tgaaataaca taaggtggtc ccgtcgatag
     2281 ccgaagctta ccgaagtata cacttaaatt cagtgcacgt ttgcttgttg agaggaaagg
     2341 ttgtgtgcgg acgaattttt ttttgaaaac attaaccctt acgtggaata aaaaaaaatg
     2401 aaatattgca aattttgctg caaagctgtg actggagtaa aattaattca cgtgccgaag
     2461 tgtgctatta agagaaaatt gtgggagcag agccttgggt gcagccttgg tgaaaactcc
     2521 caaatttgtg atacccactt taatgattcg cagtggaagg ctgcacctgc aaaaggtcag
     2581 acatttaaaa ggaggcgact caacgcagat gccgtaccta gtaaagtgat agagcctgaa
     2641 ccagaaaaga taaaagaagg ctataccagt gggagtacac aaacagagta agtttgaata
     2701 gtaaaaaaaa tcatttatgt aaacaataac gtgactgtgc gttaggtcct gttcattgtt
     2761 taatgaaaat aagagcttga gggaaaaaat tcgtactttg gagtacgaaa tgcgtcgttt
     2821 agagcagcag ccgaattaat tctagttcca gtgaaatcca agcattttct aaattaaatg
     2881 tattcttatt attatagttg ttatttttga tatatataaa caacactatt atgcccacca
     2941 tttttttgag atgcatctac acaaggaaca aacactggat gtcactttca gttcaaattg
     3001 taacgctaat cactccgaac aggtcacaaa aaattacctt aaaaagtcat aatattaaat
     3061 tagaataaat atagctgtga gggaaatata tacaaatata ttggagcaaa taaattgtac
     3121 atacaaatat ttattactaa tttctattga gacgaaatga accactcgga accatttgag
     3181 cgaaccgaat cgcgcggaac taacgacagt cgctccaagg tcgtcgaaca aaaggtgaat
     3241 gtgttgcgga gagcgggtgg gagacagcga aagagcaact acgaaacgtg gtgtggtgga
     3301 ggtgaattat gaagagggcg cgcgatttga aaagtatgta tataaaaaat atatcccggt
     3361 gttttatgta gcgataaacg agtttttgat gtaaggtatg caggtgtgta agtcttttgg
     3421 ttagaagaca aatccaaagt ctacttgtgg ggatgttcga aggggaaata cttgtattct
     3481 ataggtcata tcttgttttt attggcacaa atataattac attagctttt tgagggggca
     3541 ataaacagta aacacgatgg taataatggt aaaaaaaaaa aacaagcagt tatttcggat
     3601 atatgtcggc tactccttgc gtcgggcccg aagtcttaga gccagatatg cgagcacccg
     3661 gaagctcacg atgagaatgg ccagacccac gtagtccagc ggcagatcgg cggcggagaa
     3721 gttaagcgtc tccaggatga ccttgcccga actggggcac gtggtgttcg acgatgtgca
     3781 gctaatttcg cccggctcca cgtccgccca ttggttaatc agcagaccct cgttggcgta
     3841 acggaaccat gagaggtacg acaaccattt gaggtatact ggcaccgagc ccgagttcaa
     3901 gaagaagccg ccaaagagca ggaatggtat gataaccggc ggacccacag acagcgccat
     3961 cgaggtcgag gagctggcgc aggatattag atatccgaag gacgttgaca cattggccac
     4021 cagagtgacc agcgccaggc agttgaagaa gtgcagcact ccggcccgca gtccgatcat
     4081 cggataggca atcgccgtga agaccagtgg cactgtgaga aaaagcggca attcggcaat
     4141 cgttttgccc agaaagtatg tgtcacagcg ataaagtcga cttcgggcct ccctcataaa
     4201 aactggcagc tctgaggtga acacctaaat cgaatcgatt cattagaaag ttagtaaatt
     4261 attgaaatgc aaatgtattc taaacatgac ttacatttat cgtggcaaag acgttttgaa
     4321 aggtcatgtt ggtcaggaag aggaagatgg ctccgttgat attcatcaca cccacttgcg
     4381 tgagttgttg gcccaaaaag atgaggccaa tcaagatggc aaccatctgc aaattaaaat
     4441 gttactcgca tctcattaat attcgcgagt taaatgaaat ttatttatct tctgcaaaac
     4501 tataaactat acatctcatt gaaaaaaact aagaagggtg tggaatcagg caattctatc
     4561 taaaatctag cgaatttgtt tccaagaatt gtaagcgtta tatcatttgt ttccactgga
     4621 accactcacc gttgtctgaa taagtcgcac ttttacgagg agtggttcct tgagcaccga
     4681 cagccaggat cgccacagga ccgcccggaa ctgcatgaac caggtggcct tgtaggtgta
     4741 cccattctcc ggctgctcca gtggcttctc cagatttttg gtggccaaca actgctccat
     4801 atcccgggct actttgctaa tggcaaaatt gtcgcatatc ttggcgatcc gatcacggga
     4861 ctcgatctcc cgtccgggca caacggccaa cacctgtacg taaaagtccg ccggattgta
     4921 gttggtagga cactgggcac ccacgctgga taggagttga gatgttatgt aatactagat
     4981 acccttaata aacacatcga actcactagg aaaagaagtc gacggcttcg ctgggagtgc
     5041 ccaagaaagc taccctgccc tcggccatca gaaggatctt gtcaaagagc tcaaacagct
     5101 cggaagacgg ctgatgaatg gtcaggatga cggtcttgcc cttctgcgac agcttcttca
     5161 gcacctggac gacgctgtgg gcggtaaagg agtccagtcc ggaggtgggc tcatcgcaga
     5221 tcagaagcgg cggatcggtt agagcctcgg aggcgaatgc cagacgcttc ctttctccgc
     5281 cggacagacc tttcaccctg ccgggcacac cgatgatcgt gtgctgacat ttgctgagcg
     5341 aaagctcctg gatcacctga tccacgcggg ccactcgctg ccgataggtc agatgtcgtg
     5401 gcatccgcac catggcttgg aaaatcaggt gttccctggc cgttagggag ccgataaaga
     5461 ggtcatcctg ctggacatag gcgcacctgg cctgcatctc cttggcgtcc acaggttggc
     5521 cattgagcag tcgcatcccg gatggcgata cttggatgcc ctgcggcgat cgaaaggcaa
     5581 gggcattcag cagggtcgtc tttccggcac cggaactgcc catcacggcc aaaagttcgc
     5641 ccggataggc cacgccgcaa actgagtttc aaattggtaa ttggaccctt tattaagatt
     5701 tcacacagat cagccgactg cgaatagaaa ctcaccgttc ttgagcaaat gtttcctggg
     5761 cgccggtatg tgtcgctcgt tgcagaatag tccgcgtgtc cggttgacca gctgccgcca
     5821 tccggagccc ggctgattga ccgccccaaa gatgtccata ttgtgccagg cataggtgag
     5881 gttctcggct agttggccgc tccctgaacc ggagtcctcc ggcggactgg gtggccggag
     5941 cgtgccgtag tttttggcct gcccgaagcc ctggttaatg cagctctgcg aagccgctcc
     6001 gctgtcaccc tgcaatgata ggggatctca aatatcaact acaagcgtta tgctcatcta
     6061 accccgaaca aaaagtaccc cgaagtatcc tacgaagtag gtttatactt ttatttattt
     6121 tttgtgcatc taggatcagc ttaaaatatc tggttgttat attttttgta aaaaagaata
     6181 tagtcgaaaa tgaatgcctt tagatgtctt gatcatgata tgatctcaaa aattgtctta
     6241 tatagcgaga acagctacca gaataatctg tttcgtgtca ctatttgttt gtgcaattgc
     6301 ggtttgggat ttttgtgggt cgcagttctc acgccgcaga caatttgatg ttgcaatcgc
     6361 agttcctata gatcaagtga acttaagatg tatgcacatg tactactcac attgttcaga
     6421 tgctcggcag atgggtgttt gctgcctccg cgaattaata gctcctgatc ctcttggccc
     6481 attgccggga tttttcacac tttcccctgc ttacccaccc aaaaccaatc accaccccaa
     6541 tcactcaaaa aacaaacaaa aataagaagc gagaggagtt ttggcacagc actttgtgtt
     6601 taattgatgg cgtaaaccgc ttggagcttc gtcacgaaac cgctgacaaa atgcaactga
     6661 aggcggacat tgacgctacg taacgctaca aacggtggcg aaagagatag cggacgcagc
     6721 ggcgaaagag acggcgatat ttctgtggac agagaaggag gcaaacagcg ctgactttga
     6781 gtggaatgtc attttgagtg agaggtaatc gaaagaacct ggtacatcaa atacccttgg
     6841 atcgaagtaa atttaaaact gatcagataa gttcaatgat atccagtgca gtaaaaaaaa
     6901 aaaatgtttt ttttatctac tttccgcaaa aatgggtttt attaacttac atacatacta
     6961 gaattaggcc actagaatgc atacgcatta agcgaacatt aaaaagatgt gaaaacataa
     7021 ctattatgtc taaataaaca cacgtcagat gtatgtacgt caacggaaaa ccattgtcta
     7081 tatattacaa ttactaaata cataccaaat tgaatacata ttgatgaaaa ataataaata
     7141 ctggcgaaag caaaaaaacg aaacattttt attttattga acaactctca ggctccaggt
     7201 aggcaaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     7261 taaacttgct atgctgtttc cagcatagct cttaaacaca agaagagcct cgctcttcct
     7321 tgtgcatcgg ccgggaatcg aacccgggcc gcccgcgtgg caggcgagca ttctaccact
     7381 gaaccaccga tgcttgatgt ctacacacac tcaaagcccc gacgttaaat tgaaaatagg
     7441 tctatatata cgaactgagt ctggaaaaag aagttgagaa ttataaaaag tagtgagcac
     7501 tggcgccctc tctgcttggc gagctaacct tttcgcctct tggctgagta ggtggcgttt
     7561 cattctactc tgtaaaatta atgtagaatt gaaacactat ggtcaaaaaa tacttagggg
     7621 cataagttat aaaacgtatg aaatgaattt ttatcaacct gggctattca aaaattttcg
     7681 aattatttta tgtatttttt ttaatcgttt ttcattatag gttaaaatac actttaaaag
     7741 gaattctttc ctgtaaaata aatataaata aatatgcttt attgacagaa aatttgatgt
     7801 ttttgtattt gagtaggagc aatcacaggt gagcaaaaaa gaattcagat ccagatccac
     7861 tagagtcgac ctcgaacgtt aacgttaacg taacgttaac tcgaggcctc gaggtcgacc
     7921 tgcagccaag ctttgcgtac tcgcaaatta ttaaaaataa aactttaaaa ataatttcgt
     7981 ctaattaata ttatgagtta attcaaaccc cacggacatg ctaagggtta atcaacaatc
     8041 atatcgctgt ctcactcaga ctcaatacga cactcagaat actattcctt tcactcgcac
     8101 ttattgcaag catacgttaa gtggatgtct cttgccgacg ggaccacctt atgttatttc
     8161 atcatggtct ggccattctc atcgtgagct tccgggtgct cgcatatctg gctctaagac
     8221 ttcgggcccg acgcaaggag tagccgacat atatccgaaa taactgcttg tttttttttt
     8281 ttaccattat taccatcgtg tttactgttt attgccccct caaaaagcta atgtaattat
     8341 atttgtgcca ataaaaacaa gatatgacct atagaataca agtatttccc cttcgaacat
     8401 ccccacaagt agactttgga tttgtcttct aaccaaaaga cttacacacc tgcatacctt
     8461 acatcaaaaa ctcgtttatc gctacataaa acaccgggat atatttttta tatacatact
     8521 tttcaaatcg cgcgccctct tcataattca cctccaccac accacgtttc gtagttgctc
     8581 tttcgctgtc tcccacccgc tctccgcaac acattcacct tttgttcgac gaccttggag
     8641 cgactgtcgt tagttccgcg cgattcggtg cggtatttca caccgcatat cgacgatgta
     8701 ggtcacggtc tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc
     8761 gtactccacc tcacccatct ggtccatcat gatgaacggg tcgaggtggc ggtagttgat
     8821 cccggcgaac gcgcggcgca ccgggaagcc ctcgccctcg aaaccgctgg gcgcggtggt
     8881 cacggtgagc acgggacgtg cgacggcgtc ggcgggtgcg gatacgcggg gcagcgtcag
     8941 cgggttctcg acggtcacgg cgggcat
//
