LOCUS       sequence-331408-        7428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA targeting the yellow gene in Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  AUTHORS   Kandul NP, Belikoff EJ, Liu J, Buchman A, Li F, Yamamoto A, Yang T, 
            Shriner I, Scott MJ, Akbari OS
  TITLE     Genetically Encoded CRISPR Components Yield Efficient Gene Editing 
            in the Invasive Pest Drosophila suzukii.
  JOURNAL   CRISPR J. 2021 Oct;4(5):739-751. doi: 10.1089/crispr.2021.0032.
  PUBMED    34661429
REFERENCE   2  (bases 1 to 7428)
  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: 
            "10.1089/crispr.2021"; journalName: "CRISPR J"; date: "2021-10"; 
            volume: "4"; issue: "5"; pages: "739-751"
COMMENT     Sequence Label: gRNAy
FEATURES             Location/Qualifiers
     source          1..7428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      793..802
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(799..801,802..804,805..1518)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(799..801,802..804,805..1515)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1641..1762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1796..2215
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        2236..2311
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(2894..2896,2897..2899,2900..3613)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    3735..3856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3928..4027
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     repeat_region   4815..4849
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5030..5046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5064..5098
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5279..5295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5303..5319
                     /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(5327..5333,5334..5351,5352..5357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5372..5393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5510..5527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5681..6269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5761..5780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6440..7231,7232..7300))
                     /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     7063..7082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7301..7405)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttcatttggc aaaatattag agatattatc gcgcaaatat ctcttcaaag taggagcttc
       61 taaacgctta cgcataaacg atgacgtcag gctcatgtaa aggtttctca taaatttttt
      121 gcgactttga accttttctc ccttgctact gacattatgg ctgtatataa taaaagaatt
      181 tatgcaggca atgtttatca ttccgtacaa taatgccata ggccacctat tcgtcttcct
      241 actgcaggtc atcacagaac acatttggtc tagcgtgtcc actccgcctt tagtttgatt
      301 ataatacata accatttgcg gtttaccggt actttcgttg atagaagcat cctcatcaca
      361 agatgataat aagtatacca tcttagctgg cttcggttta tatgagacga gagtaagggg
      421 tccgtcaaaa caaaacatcg atgttcccac tggcctggag cgactgtttt tcagtacttc
      481 cggtatctcg cgtttgtttg atcgcacggt tcccacaatg gttaattcga gctcgcccgg
      541 ggatctaatt caattagaga ctaattcaat tagagctaat tcaattagga tccaagctta
      601 tcgatttcga accctcgacc gccggagtat aaatagaggc gcttcgtcta cggagcgaca
      661 attcaattca aacaagcaaa gtgaacacgt cgctaagcga aagctaagca aataaacaag
      721 cgcagctgaa caagctaaac aatcggggta ccgctagagt cgacggtacc gcgggcccgg
      781 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      841 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      901 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      961 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1021 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1081 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1141 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1201 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1261 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1321 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1381 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1441 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1501 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg
     1561 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     1621 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     1681 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1741 ccaaactcat caatgtatct taaagcttat cgatacgcgt acggagcggc gcgccttttt
     1801 tgctcacctg tgattgctcc tactcaaata caaaaacatc aaattttctg tcaataaagc
     1861 atatttattt atatttattt tacaggaaag aattcctttt aaagtgtatt ttaacctata
     1921 atgaaaaacg attaaaaaaa atacataaaa taattcgaaa atttttgaat agcccaggtt
     1981 gataaaaatt catttcatac gttttataac ttatgcccct aagtattttt tgaccatagt
     2041 gtttcaattc tacattaatt ttacagagta gaatgaaacg ccacctactc agccaagagg
     2101 cgaaaaggtt agctcgccaa gcagagaggg cgccagtgct cactactttt tataattctc
     2161 aacttctttt tccagactca gttcgtatat atagacctat ttcaatttaa cgtcgggtcc
     2221 tggacaccgg aaccggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2281 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt taaaccatac tcggtggcct
     2341 ccccaccaca ctcttttgcc cccccaccac caactttttt gcactgcaaa aaaacacgct
     2401 tttgcacgcg ggcccataca tagtacaaac tctacgtttc gtagactatt ttacataaat
     2461 agtctacacc gttgtatacg ctccaaatac actaccacac attgaacctt tttgcagtgc
     2521 aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc gggtcgcgtc ctgtcacgta
     2581 cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt gacaggacgc cagcttcctg
     2641 tgttgctaac cgcagccgga cgcaactcct tatcggaaca ggacgcgcct ccatatcagc
     2701 cgcgcgttat ctcatgcgcg tgaccggaca cgaggcgccc gtcccgctta tcgcgcctat
     2761 aaatacagcc cgcaacgatc tggtaaacac agttgaacag catctgttac agcgacacaa
     2821 catgagccgg tccaacaacg ccaacgcgcc cacgccatcc aaccgccgcc gcaacctgtc
     2881 tctggtgctc gagatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2941 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3001 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3061 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc
     3121 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3181 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3241 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3301 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga
     3361 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag
     3421 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3481 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3541 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     3601 gctgtacaag taagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     3661 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3721 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3781 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3841 tcatcaatgt atcttaaagc ttgtactgac ggacacaccg aagccccggc ggcaaccctc
     3901 agcggatgcc ccggggcttc acgttttccc aggtcagaag cggttttcgg gagtagtgcc
     3961 ccaactgggg taacctttga gttctctcag ttgggggcgt agggtcgccg acatgacaca
     4021 aggggttgtg accggggtgg acacgtacgc gggtgcttac gaccgtcagt cgcgcgagcg
     4081 cgaccgaatt cgaatggcca tgggacgtcg acctgaggta attataaccc gggccctata
     4141 tatggatcca attgcaatga tcatcatgac agatctgaca atgttcagtg cagagactcg
     4201 gctacgcctc gtggactttg aagttgacca acaatgttta ttcttacctc taatagtcct
     4261 ctgtggcaag gtcaagattc tgttagaagc caatgaagaa cctggttgtt caataacatt
     4321 ttgttcgtct aatatttcac taccgcttga cgttggctgc acttcatgta cctcatctat
     4381 aaacgcttct tctgtatcgc tctggacgtc atcttcactt acgtgatctg atatttcact
     4441 gtcagaatcc tcaccaacaa gctcgtcatc gctttgcaga agagcagaga ggatatgctc
     4501 atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata acaagaaaat
     4561 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga
     4621 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt
     4681 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc
     4741 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca
     4801 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taaaaaagat
     4861 ttgcgcttta ctcgacctaa actttaaaca cgtcatagaa tcttcgtttg acaaaaacca
     4921 cattgtggcc aagctgtgtg acgcgacgcg cgctaaagaa tggcaaacca agtcgcgcga
     4981 gcgtcgactc tagaggatcc ccgggtaccg agctcgaatt cgtaatcatg tcatagctgt
     5041 ttcctgtgtg aaattgyaga atgcatgcgt caattttacg cagactatct ttctagggtt
     5101 aaaaaagatt tgcgctttac tcgacctaaa ctttaaacac gtcatagaat cttcgtttga
     5161 caaaaaccac attgtggcca agctgtgtga cgcgacgcgc gctaaagaat ggcaaaccaa
     5221 gtcgcgcgag cgtcgactct agaggatccc cgggtaccga gctcgaattc gtaatcatgt
     5281 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5341 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7381 aaaataaaca aataggggtt ccgcgcagtc atctgatgta ccaggcac
//
