LOCUS       sequence-158774-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drosophila gRNA expression plasmid targets ebony.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  AUTHORS   Kane NS, Vora M, Varre KJ, Padgett RW
  TITLE     Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila 
            Using a Co-CRISPR Strategy.
  JOURNAL   G3 (Bethesda). 2017 Jan 5;7(1):87-93. doi: 10.1534/g3.116.036723.
  PUBMED    27793971
REFERENCE   2  (bases 1 to 6249)
  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: "10.1534/g3.116"; 
            journalName: "G3 (Bethesda)"; date: "2017-01-5- 5"; volume: "7"; 
            issue: "1"; pages: "87-93"
COMMENT     Sequence Label: pCFD3-ebony
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..427
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        447..522
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(833..853)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(833..851)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(872..888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(872..888)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(885..907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    896..912
                     /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(920..926,927..944,945..950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    965..986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1103..1120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1274..1862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1354..1373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2033..2824,2825..2893))
                     /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     2656..2675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2894..2998)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3024..3479)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3161..3182)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3373..3392
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3606..3628
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3620..3637
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3621..3637
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3644..3663
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3644..3662
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3854..3971,4048..4141,4233..4839,4894..5027,
                     5088..5248,5322..5347))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the brown 
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    5662..5731
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
       61 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
      121 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
      181 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
      241 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
      301 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
      361 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
      421 aacgtcgcca caattgtcga tcgtcagttt tagagctaga aatagcaagt taaaataagg
      481 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgc ctacctggag
      541 cctgagagtt gttcaataaa ataaaaatgt ttcgtttttt tgctttcgcc agtatttatt
      601 atttttcatc aatatgtatt caatttggta tgtatttagt aattgtaata tatagacaat
      661 ggttttccgt tgacgtacat acatctgacg tgtgtttatt tagacataat agttatgttt
      721 tcacatcttt ttaatgttcg cttaatgcgt atgcattcta gacaattgtg ctcggcaaca
      781 gtatatttgt ggtgtgccaa ccaacaacct gcaggagctc cagcttttgt tccctttagt
      841 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      901 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      961 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1081 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1141 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1201 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1261 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1321 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1381 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1441 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1501 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1561 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1621 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1681 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     1741 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1801 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1861 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1921 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1981 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2041 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2101 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2161 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2221 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2281 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     2341 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     2401 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2461 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2521 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2581 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2641 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     2701 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     2761 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     2821 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     2881 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3001 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     3061 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3121 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3181 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3241 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3301 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3361 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3421 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3481 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3541 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3601 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
     3661 gggcgaattg ggtacaagct tggatttatt ttgttatgtt atatgtatta tatgtcagac
     3721 ataaagaaaa ggaacacatc aaatgtgata acaaagacta aacaagtaat tttattacac
     3781 caaaacgaca aaacagtagg cagaacaaac aacgcatagc caaacattga cgaattggat
     3841 accctgccga ttgtcagaca cttttgttga tcagtttctt gcgaatggtc tcgtccagcg
     3901 gtggaatcgc ctcgcgggga atcagaaaag tggacagatt gaacagatcc agaaacacct
     3961 tgtaccgatc actgaaacca aaaaaaaaca aagggagaac agtttgagtt cattgatccc
     4021 cgatataatc acatctgcga tgatcacctg agagtggagc gcagatattg atatccagac
     4081 gagccaccag tgcccaactg ttgggatcca atcatgcgtt gcaccatgat cacgtgattg
     4141 tctgcggcgg gaatagaaag tatttggtta ggaaaaccag tcttaaacat aagatatatt
     4201 tataaaagag tatcaaagaa tgcaatactt acatctccac ttggttatta acgagtcgat
     4261 gtccatgagc agggtgagca actggtgtgg ttggctgaac ctgggttcat ccctatagaa
     4321 ggtgatcatg atggctccct gaagggcacg atggctaaac cggcgatccc cacgacgcac
     4381 cagtgcatcg tgcactgccg gatcaaagat ggagcgatac acctcgcgtc gcttctcaat
     4441 gtccatgagg cggtagtttt tcgccttctc cacgggctcc tccatggcgc tctgtacctg
     4501 cgcctccagg aatcgatcga cgctctcctg aaacttggcc cagaagttga agccactctc
     4561 ctccagtccg ggcgtcctct ccagccatcg ctgcactagc tccagtagcg agggatcttt
     4621 ctccgagttg cgaatcgagt tccgcgcctc ctcgtcgcta aagacatccg agtacttctg
     4681 gttgtatctc acccgctgct ctgtcagaac tcccagcttg ttctcgatca aacggaactg
     4741 cagcgactga aaaccagatg cgggtgccag gtacttgcgg aagtccatga agtctagcgg
     4801 ggtcatggtc tccagaatgg gcacttggtc caccaggagc tgtacaaagg aagttataaa
     4861 cggattttgg taagagattc agaaagcact cacttttaga atcagaacca ctcggttcag
     4921 tcgcttgaca atctccagcg tcttggtttc atcgatgacc tctgcatcca acatgtctcg
     4981 tatggagtcg aactcaaaga tgatctgctt gaaccaaagc tcgtaggctg tggcgaaggt
     5041 acttaaatgc cattgagtgt tgtcatcaaa gttgtaaacc tactcaccct ggtgcgtgat
     5101 gatgaacaga tgctcatcgt gcacgggtcg cttgtcctcc tcggacagca tacactgggc
     5161 atccagcagt ttgtccagca tcagatactc tccatagatt ttgcccactt ccgtggttaa
     5221 tggcaccgcc gaatcatcgt gatcgtttct gtatgggttt gaattgaatc gcagaactga
     5281 agatcgattg gcattcctgg acagcacgtg ctggtgctca cccgtttcct gcatagggac
     5341 agctcatggt gcacagctca gatcagatcg tgactcctcg agcggcggat gctggcgaac
     5401 tgatctccgc cagcggaccg gagatgagac cccagcgaac cgataacaga gcgagagagc
     5461 tccagttccg actgattgca cagtcggtga tctgggcgat gggcactgcc agataggctg
     5521 ggaattatca atcacttgag gtgaaagtgc ggcgcacaca aatccaagct tgatatcatc
     5581 gatctcgagg ctgcatccaa cgcgttggga gctctccgga tcaattcggc ttcaggtacc
     5641 gtcgacgatg taggtcacgg tctcgaagcc gcggtgcggg tgccagggcg tgcccttggg
     5701 ctccccgggc gcgtactcca cctcacccat ctggtccatc atgatgaacg ggtcgaggtg
     5761 gcggtagttg atcccggcga acgcgcggcg caccgggaag ccctcgccct cgaaaccgct
     5821 gggcgcggtg gtcacggtga gcacgggacg tgcgacggcg tcggctgggt gcggatacgc
     5881 ggggcagcgt cagcgggttc tcgacggtca cggcgggcat gtcgacaagc cgaattgatc
     5941 cactagaagg cctaattcgg tacactagtt ggccacgtaa taagtgtgcg ttgaatttat
     6001 tcgcaaaaac attgcatatt ttcggcaaag taaaattttg ttgcatacct tatcaaaaaa
     6061 taagtgctgc atacttttta gagaaaccaa ataatttttt attgcatacc cgtttttaat
     6121 aaaatacatt gcataccctc ttttaataaa aaatattgca tactttgacg aaacaaattt
     6181 tcgttgcata cccaataaaa gattattata ttgcataccc gtttttaata aaatacattg
     6241 catagatct
//
