LOCUS       sequence-208995-        6250 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 6250)
  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 6250)
  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..6250
                     /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        448..523
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(834..854)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(834..852)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(873..889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(873..889)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(886..908)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    897..913
                     /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(921..927,928..945,946..951))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    966..987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1104..1121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1275..1863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1355..1374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2034..2825,2826..2894))
                     /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     2657..2676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2895..2999)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3025..3480)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3162..3183)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3374..3393
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3607..3629
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3621..3638
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3622..3638
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3645..3664
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3645..3663
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3855..3972,4049..4142,4234..4840,4895..5028,
                     5089..5249,5323..5348))
                     /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    5663..5732
                     /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 aacgtcgaaa aagagatgcc cgcagaggtt ttagagctag aaatagcaag ttaaaataag
      481 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg cctacctgga
      541 gcctgagagt tgttcaataa aataaaaatg tttcgttttt ttgctttcgc cagtatttat
      601 tatttttcat caatatgtat tcaatttggt atgtatttag taattgtaat atatagacaa
      661 tggttttccg ttgacgtaca tacatctgac gtgtgtttat ttagacataa tagttatgtt
      721 ttcacatctt tttaatgttc gcttaatgcg tatgcattct agacaattgt gctcggcaac
      781 agtatatttg tggtgtgcca accaacaacc tgcaggagct ccagcttttg ttccctttag
      841 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      901 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      961 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1021 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1081 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1681 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3001 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     3061 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3121 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3181 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3241 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3301 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3361 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3421 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3481 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     3541 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     3601 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     3661 agggcgaatt gggtacaagc ttggatttat tttgttatgt tatatgtatt atatgtcaga
     3721 cataaagaaa aggaacacat caaatgtgat aacaaagact aaacaagtaa ttttattaca
     3781 ccaaaacgac aaaacagtag gcagaacaaa caacgcatag ccaaacattg acgaattgga
     3841 taccctgccg attgtcagac acttttgttg atcagtttct tgcgaatggt ctcgtccagc
     3901 ggtggaatcg cctcgcgggg aatcagaaaa gtggacagat tgaacagatc cagaaacacc
     3961 ttgtaccgat cactgaaacc aaaaaaaaac aaagggagaa cagtttgagt tcattgatcc
     4021 ccgatataat cacatctgcg atgatcacct gagagtggag cgcagatatt gatatccaga
     4081 cgagccacca gtgcccaact gttgggatcc aatcatgcgt tgcaccatga tcacgtgatt
     4141 gtctgcggcg ggaatagaaa gtatttggtt aggaaaacca gtcttaaaca taagatatat
     4201 ttataaaaga gtatcaaaga atgcaatact tacatctcca cttggttatt aacgagtcga
     4261 tgtccatgag cagggtgagc aactggtgtg gttggctgaa cctgggttca tccctataga
     4321 aggtgatcat gatggctccc tgaagggcac gatggctaaa ccggcgatcc ccacgacgca
     4381 ccagtgcatc gtgcactgcc ggatcaaaga tggagcgata cacctcgcgt cgcttctcaa
     4441 tgtccatgag gcggtagttt ttcgccttct ccacgggctc ctccatggcg ctctgtacct
     4501 gcgcctccag gaatcgatcg acgctctcct gaaacttggc ccagaagttg aagccactct
     4561 cctccagtcc gggcgtcctc tccagccatc gctgcactag ctccagtagc gagggatctt
     4621 tctccgagtt gcgaatcgag ttccgcgcct cctcgtcgct aaagacatcc gagtacttct
     4681 ggttgtatct cacccgctgc tctgtcagaa ctcccagctt gttctcgatc aaacggaact
     4741 gcagcgactg aaaaccagat gcgggtgcca ggtacttgcg gaagtccatg aagtctagcg
     4801 gggtcatggt ctccagaatg ggcacttggt ccaccaggag ctgtacaaag gaagttataa
     4861 acggattttg gtaagagatt cagaaagcac tcacttttag aatcagaacc actcggttca
     4921 gtcgcttgac aatctccagc gtcttggttt catcgatgac ctctgcatcc aacatgtctc
     4981 gtatggagtc gaactcaaag atgatctgct tgaaccaaag ctcgtaggct gtggcgaagg
     5041 tacttaaatg ccattgagtg ttgtcatcaa agttgtaaac ctactcaccc tggtgcgtga
     5101 tgatgaacag atgctcatcg tgcacgggtc gcttgtcctc ctcggacagc atacactggg
     5161 catccagcag tttgtccagc atcagatact ctccatagat tttgcccact tccgtggtta
     5221 atggcaccgc cgaatcatcg tgatcgtttc tgtatgggtt tgaattgaat cgcagaactg
     5281 aagatcgatt ggcattcctg gacagcacgt gctggtgctc acccgtttcc tgcataggga
     5341 cagctcatgg tgcacagctc agatcagatc gtgactcctc gagcggcgga tgctggcgaa
     5401 ctgatctccg ccagcggacc ggagatgaga ccccagcgaa ccgataacag agcgagagag
     5461 ctccagttcc gactgattgc acagtcggtg atctgggcga tgggcactgc cagataggct
     5521 gggaattatc aatcacttga ggtgaaagtg cggcgcacac aaatccaagc ttgatatcat
     5581 cgatctcgag gctgcatcca acgcgttggg agctctccgg atcaattcgg cttcaggtac
     5641 cgtcgacgat gtaggtcacg gtctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg
     5701 gctccccggg cgcgtactcc acctcaccca tctggtccat catgatgaac gggtcgaggt
     5761 ggcggtagtt gatcccggcg aacgcgcggc gcaccgggaa gccctcgccc tcgaaaccgc
     5821 tgggcgcggt ggtcacggtg agcacgggac gtgcgacggc gtcggctggg tgcggatacg
     5881 cggggcagcg tcagcgggtt ctcgacggtc acggcgggca tgtcgacaag ccgaattgat
     5941 ccactagaag gcctaattcg gtacactagt tggccacgta ataagtgtgc gttgaattta
     6001 ttcgcaaaaa cattgcatat tttcggcaaa gtaaaatttt gttgcatacc ttatcaaaaa
     6061 ataagtgctg catacttttt agagaaacca aataattttt tattgcatac ccgtttttaa
     6121 taaaatacat tgcataccct cttttaataa aaaatattgc atactttgac gaaacaaatt
     6181 ttcgttgcat acccaataaa agattattat attgcatacc cgtttttaat aaaatacatt
     6241 gcatagatct
//
