LOCUS       sequence-211391-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses U6:3-sgRNA targeting ovoD1 mutation in Drosophila 
            melanogaster.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6370)
  AUTHORS   Ewen-Campen B, Perrimon N
  TITLE     ovo(D) Co-selection: A Method for Enriching CRISPR/Cas9-Edited 
            Alleles in Drosophila.
  JOURNAL   G3 (Bethesda). 2018 Jul 31;8(8):2749-2756. doi: 
            10.1534/g3.118.200498.
  PUBMED    29934375
REFERENCE   2  (bases 1 to 6370)
  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/g3.118"; 
            journalName: "G3 (Bethesda)"; date: "2018-07-31- 31"; volume: "8"; 
            issue: "8"; pages: "2749-2756"
COMMENT     Sequence Label: pCFD3-ovoD1
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..70
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    312..697
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        711..1131
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1152..1227
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1538..1558)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1538..1556)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1577..1593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1577..1593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1590..1612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1601..1617
                     /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(1625..1631,1632..1649,1650..1655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1670..1691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1808..1825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1979..2567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2059..2078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2738..3529,3530..3598))
                     /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     3361..3380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3599..3703)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3730..4186
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3817..3836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4027..4048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4312..4334
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4326..4343
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4327..4343
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4353..4372
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4353..4371
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4563..4680,4757..4850,4942..5548,5603..5736,
                     5797..5957,6031..6056))
                     /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"
ORIGIN
        1 ctcgaagccg cggtgcgggt gccagggcgt gcccttgggc tccccgggcg cgtactccac
       61 ctcacccatc tggtccatca tgatgaacgg gtcgaggtgg cggtagttga tcccggcgaa
      121 cgcgcggcgc accgggaagc cctcgccctc gaaaccgctg ggcgcggtgg tcacggtgag
      181 cacgggacgt gcgacggcgt cggctggtgc ggatacgcgg ggcagcgtca gcgggttctc
      241 gacggtcacg gcgggcatgt cgacaagccg aattgatcca ctagaaggcc taattcggta
      301 cactagttgg ccacgtaata agtgtgcgtt gaatttattc gcaaaaacat tgcatatttt
      361 cggcaaagta aaattttgtt gcatacctta tcaaaaaata agtgctgcat actttttaga
      421 gaaaccaaat aattttttat tgcatacccg tttttaataa aatacattgc ataccctctt
      481 ttaataaaaa atattgcata ctttgacgaa acaaattttc gttgcatacc caataaaaga
      541 ttattatatt gcatacccgt ttttaataaa atacattgca taccctcttt taataaagaa
      601 tattgcatac gttgacgaaa caaattttcg ttgcataccc aataaaagat tattatattg
      661 catacctttt cttgccatac catttagccg atcaattgag atctgaattc ttttttgctc
      721 acctgtgatt gctcctactc aaatacaaaa acatcaaatt ttctgtcaat aaagcatatt
      781 tatttatatt tattttacag gaaagaattc cttttaaagt gtattttaac ctataatgaa
      841 aaacgattaa aaaaaataca taaaataatt cgaaaatttt tgaatagccc aggttgataa
      901 aaattcattt catacgtttt ataacttatg cccctaagta ttttttgacc atagtgtttc
      961 aattctacat taattttaca gagtagaatg aaacgccacc tactcagcca agaggcgaaa
     1021 aggttagctc gccaagcaga gagggcgcca gtgctcacta ctttttataa ttctcaactt
     1081 ctttttccag actcagttcg tatatataga cctattttca atttaacgtc gaaaaagaga
     1141 tgcccgcaga ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     1201 cttgaaaaag tggcaccgag tcggtgcttt tttgcctacc tggagcctga gagttgttca
     1261 ataaaataaa aatgtttcgt ttttttgctt tcgccagtat ttattatttt tcatcaatat
     1321 gtattcaatt tggtatgtat ttagtaattg taatatatag acaatggttt tccgttgacg
     1381 tacatacatc tgacgtgtgt ttatttagac ataatagtta tgttttcaca tctttttaat
     1441 gttcgcttaa tgcgtatgca ttctagacaa ttgtgctcgg caacagtata tttgtggtgt
     1501 gccaaccaac aacctgcagg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     1561 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1681 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2341 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2401 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3721 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3781 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3841 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3901 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3961 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4021 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4081 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaaatga gctgatttaa
     4141 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat tcgccattca
     4201 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     4261 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     4321 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
     4381 gtacaagctt ggatttattt tgttatgtta tatgtattat atgtcagaca taaagaaaag
     4441 gaacacatca aatgtgataa caaagactaa acaagtaatt ttattacacc aaaacgacaa
     4501 aacagtaggc agaacaaaca acgcatagcc aaacattgac gaattggata ccctgccgat
     4561 tgtcagacac ttttgttgat cagtttcttg cgaatggtct cgtccagcgg tggaatcgcc
     4621 tcgcggggaa tcagaaaagt ggacagattg aacagatcca gaaacacctt gtaccgatca
     4681 ctgaaaccaa aaaaaaacaa agggagaaca gtttgagttc attgatcccc gatataatca
     4741 catctgcgat gatcacctga gagtggagcg cagatattga tatccagacg agccaccagt
     4801 gcccaactgt tgggatccaa tcatgcgttg caccatgatc acgtgattgt ctgcggcggg
     4861 aatagaaagt atttggttag gaaaaccagt cttaaacata agatatattt ataaaagagt
     4921 atcaaagaat gcaatactta catctccact tggttattaa cgagtcgatg tccatgagca
     4981 gggtgagcaa ctggtgtggt tggctgaacc tgggttcatc cctatagaag gtgatcatga
     5041 tggctccctg aagggcacga tggctaaacc ggcgatcccc acgacgcacc agtgcatcgt
     5101 gcactgccgg atcaaagatg gagcgataca cctcgcgtcg cttctcaatg tccatgaggc
     5161 ggtagttttt cgccttctcc acgggctcct ccatggcgct ctgtacctgc gcctccagga
     5221 atcgatcgac gctctcctga aacttggccc agaagttgaa gccactctcc tccagtccgg
     5281 gcgtcctctc cagccatcgc tgcactagct ccagtagcga gggatctttc tccgagttgc
     5341 gaatcgagtt ccgcgcctcc tcgtcgctaa agacatccga gtacttctgg ttgtatctca
     5401 cccgctgctc tgtcagaact cccagcttgt tctcgatcaa acggaactgc agcgactgaa
     5461 aaccagatgc gggtgccagg tacttgcgga agtccatgaa gtctagcggg gtcatggtct
     5521 ccagaatggg cacttggtcc accaggagct gtacaaagga agttataaac ggattttggt
     5581 aagagattca gaaagcactc acttttagaa tcagaaccac tcggttcagt cgcttgacaa
     5641 tctccagcgt cttggtttca tcgatgacct ctgcatccaa catgtctcgt atggagtcga
     5701 actcaaagat gatctgcttg aaccaaagct cgtaggctgt ggcgaaggta cttaaatgcc
     5761 attgagtgtt gtcatcaaag ttgtaaacct actcaccctg gtgcgtgatg atgaacagat
     5821 gctcatcgtg cacgggtcgc ttgtcctcct cggacagcat acactgggca tccagcagtt
     5881 tgtccagcat cagatactct ccatagattt tgcccacttc cgtggttaat ggcaccgccg
     5941 aatcatcgtg atcgtttctg tatgggtttg aattgaatcg cagaactgaa gatcgattgg
     6001 cattcctgga cagcacgtgc tggtgctcac ccgtttcctg catagggaca gctcatggtg
     6061 cacagctcag atcagatcgt gactcctcga gcggcggatg ctggcgaact gatctccgcc
     6121 agcggaccgg agatgagacc ccagcgaacc gataacagag cgagagagct ccagttccga
     6181 ctgattgcac agtcggtgat ctgggcgatg ggcactgcca gataggctgg gaattatcaa
     6241 tcacttgagg tgaaagtgcg gcgcacacaa atccaagctt gatatcatcg atctcgaggc
     6301 tgcatccaac gcgttgggag ctctccggat caattcggct tcaggtaccg tcgacgatgt
     6361 aggtcacggt
//
