LOCUS       sequence-242548-        7999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression plasmid for CRISPR mutagenesis in Drosophila (with 
            mini-white transformation marker)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  TITLE     Insect expression, CRISPR
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7999)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCFD3.1-w-dU6:3gRNA
FEATURES             Location/Qualifiers
     source          1..7999
                     /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        446..521
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(832..852)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(832..850)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(871..887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(871..887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(884..906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    895..911
                     /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(919..925,926..943,944..949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    964..985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1102..1119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1273..1861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1353..1372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2032..2823,2824..2892))
                     /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     2655..2674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2893..2997)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3024..3480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3111..3130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3321..3342
                     /label=F1ori-F
                     /note="F1 origin, forward 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     3647..3666
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3647..3665
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3705..3723)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(4175..4246,4648..4921,4996..5650,5712..6027,6231..6362,
                     6433..7047)
                     /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"
     protein_bind    7296..7365
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    7607..7992
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
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 aacgtcgggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt aaaataaggc
      481 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgcc tacctggagc
      541 ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca gtatttatta
      601 tttttcatca atatgtattc aatttggtat gtatttagta attgtaatat atagacaatg
      661 gttttccgtt gacgtacata catctgacgt gtgtttattt agacataata gttatgtttt
      721 cacatctttt taatgttcgc ttaatgcgta tgcattctag acaattgtgc tcggcaacag
      781 tatatttgtg gtgtgccaac caacaacctg caggagctcc agcttttgtt ccctttagtg
      841 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      901 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      961 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1081 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1681 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3001 atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3361 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3421 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3481 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3541 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3601 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     3661 atagggcgaa ttgggtacaa gcttgtaccg ggcccctagt atgtatgtaa gttaataaaa
     3721 cccatttttg cggaaagtag ataaaaaaaa catttttttt ttttactgca ctggatatca
     3781 ttgaacttat ctgatcagtt ttaaatttac ttcgatccaa gggtatttga tgtaccaggt
     3841 tctttcgatt acctctcact caaaatgaca ttccactcaa agtcagcgct gtttgcctcc
     3901 ttctctgtcc acagaaatat cgccgtctct ttcgccgctg cgtccgctat ctctttcgcc
     3961 accgtttgta gcgttacgta gcgtcaatgt ccgccttcag ttgcattttg tcagcggttt
     4021 cgtgacgaag ctccaagcgg tttacgccat caattaaaca caaagtgctg tgccaaaact
     4081 cctctcgctt cttatttttg tttgtttttt gagtgattgg ggtggtgatt ggttttgggt
     4141 gggtaagcag gggaaagtgt gaaaaatccc ggcaatgggc caagaggatc aggagctatt
     4201 aattcgcgga ggcagcaaac acccatctgc cgagcatctg aacaatgtga gtagtacatg
     4261 tgcatacatc ttaagttcac ttgatctata ggaactgcga ttgcaacatc aaattgtctg
     4321 cggcgtgaga actgcgaccc acaaaaatcc caaaccgcaa ttgcacaaac aaatagtgac
     4381 acgaaacaga ttattctggt agctgttctc gctatataag acaatttttg agatcatatc
     4441 atgatcaaga catctaaagg cattcatttt cgactatatt cttttttaca aaaaatataa
     4501 caaccagata ttttaagctg atcctagatg cacaaaaaat aaataaaagt ataaacctac
     4561 ttcgtaggat acttcggggt actttttgtt cggggttaga tgagcataac gcttgtagtt
     4621 gatatttgag atcccctatc attgcagggt gacagcggag cggcttcgca gagctgcatt
     4681 aaccagggct tcgggcaggc caaaaactac ggcacgctcc ggccacccag tccgccggag
     4741 gactccggtt cagggagcgg ccaactagcc gagaacctca cctatgcctg gcacaatatg
     4801 gacatctttg gggcggtcaa tcagccgggc tccggatggc ggcagctggt caaccggaca
     4861 cgcggactat tctgcaacga gcgacacata ccggcgccca ggaaacattt gctcaagaac
     4921 ggtgagtttc tattcgcagt cggctgatct gtgtgaaatc ttaataaagg gtccaattac
     4981 caatttgaaa ctcagtttgc ggcgtggcct atccgggcga acttttggcc gtgatgggca
     5041 gttccggtgc cggaaagacg accctgctga atgcccttgc ctttcgatcg ccgcagggca
     5101 tccaagtatc gccatccggg atgcgactgc tcaatggcca acctgtggac gccaaggaga
     5161 tgcaggccag gtgcgcctat gtccagcagg atgacctctt tatcggctcc ctaacggcca
     5221 gggaacacct gattttccaa gccatggtgc ggatgccacg acatctgacc tatcggcagc
     5281 gagtggcccg cgtggatcag gtgatccagg agctttcgct cagcaaatgt cagcacacga
     5341 tcatcggtgt gcccggcagg gtgaaaggtc tgtccggcgg agaaaggaag cgtctggcat
     5401 tcgcctccga ggctctaacc gatccgccgc ttctgatctg cgatgagccc acctccggac
     5461 tggactcctt taccgcccac agcgtcgtcc aggtgctgaa gaagctgtcg cagaagggca
     5521 agaccgtcat cctgaccatt catcagccgt catccgagct gtttgagctc tttgacaaga
     5581 tccttctgat ggccgagggc agggtagctt tcttgggcac tcccagcgaa gccgtcgact
     5641 tcttttccta gtgagttcga tgtgtttatt aagggtatct agtattacat aacatctcaa
     5701 ctcctatcca gcgtgggtgc ccagtgtcct accaactaca atccggcgga cttttacgta
     5761 caggtgttgg ccgttgtgcc cggacgggag atcgagtccc gtgatcggat cgccaagata
     5821 tgcgacaatt ttgccattag caaagtagcc cgggatatgg agcagttgtt ggccaccaaa
     5881 aatctggaga agccactgga gcagccggag aatgggtaca cctacaaggc cacctggttc
     5941 atgcagttcc gggcggtcct gtggcgatcc tggctgtcgg tgctcaagga accactcctc
     6001 gtaaaagtgc gacttattca gacaacggtg agtggttcca gtggaaacaa atgatataac
     6061 gcttacaatt cttggaaaca aattcgctag attttagata gaattgcctg attccacacc
     6121 cttcttagtt tttttcaatg agatgtatag tttatagttt tgcagaagat aaataaattt
     6181 catttaactc gcgaatatta atgagatgcg agtaacattt taatttgcag atggttgcca
     6241 tcttgattgg cctcatcttt ttgggccaac aactcacgca agtgggtgtg atgaatatca
     6301 acggagccat cttcctcttc ctgaccaaca tgacctttca aaacgtcttt gccacgataa
     6361 atgtaagtca tgtttagaat acatttgcat ttcaataatt tactaacttt ctaatgaatc
     6421 gattcgattt aggtgttcac ctcagagctg ccagttttta tgagggaggc ccgaagtcga
     6481 ctttatcgct gtgacacata ctttctgggc aaaacgattg ccgaattgcc gctttttctc
     6541 acagtgccac tggttttcac ggcgattgcc tatccgatga tcggactgcg ggccggagtg
     6601 ctgcacttct tcaactgcct ggcgctggtc actctggtgg ccaatgtgtc aacgtccttc
     6661 ggatatctaa tatcctgcgc cagctcctcg acctcgatgg cgctgtctgt gggtccgccg
     6721 gttatcatac cattcctgct ctttggcggc ttcttcttga actcgggctc ggtgccagta
     6781 tacctcaaat ggttgtcgta cctctcatgg ttccgttacg ccaacgaggg tctgctgatt
     6841 aaccaatggg cggacgtgga gccgggcgaa attagctgca catcgtcgaa caccacgtgc
     6901 cccagttcgg gcaaggtcat cctggagacg cttaacttct ccgccgccga tctgccgctg
     6961 gactacgtgg gtctggccat tctcatcgtg agcttccggg tgctcgcata tctggctcta
     7021 agacttcggg cccgacgcaa ggagtagccg acatatatcc gaaataactg cttgtttttt
     7081 tttttaccat tattaccatc gtgtttactg tttattgccc cctcaaaaag ctaatgtaat
     7141 tatatttgtg ccaataaaaa caagatatga cctatagaat acaagtattt ccccttcgaa
     7201 catccccaca agtagacttt ggatttgttt ctaaccaaaa gacttacaca cctgcatacc
     7261 ttagaattgg taccgtcgac gatgtaggtc acggtctcga agccgcggtg cgggtgccag
     7321 ggcgtgccct tgggctcccc gggcgcgtac tccacctcac ccatctggtc catcatgatg
     7381 aacgggtcga ggtggcggta gttgatcccg gcgaacgcgc ggcgcaccgg gaagccctcg
     7441 ccctcgaaac cgctgggcgc ggtggtcacg gtgagcacgg gacgtgcgac ggcgtcggct
     7501 ggtgcggata cgcggggcag cgtcagcggg ttctcgacgg tcacggcggg catgtcgaca
     7561 agccgaattg atccactaga aggcctaatt cggtacacta gttggccacg taataagtgt
     7621 gcgttgaatt tattcgcaaa aacattgcat attttcggca aagtaaaatt ttgttgcata
     7681 ccttatcaaa aaataagtgc tgcatacttt ttagagaaac caaataattt tttattgcat
     7741 acccgttttt aataaaatac attgcatacc ctcttttaat aaaaaatatt gcatactttg
     7801 acgaaacaaa ttttcgttgc atacccaata aaagattatt atattgcata cccgttttta
     7861 ataaaataca ttgcataccc tcttttaata aagaatattg catacgttga cgaaacaaat
     7921 tttcgttgca tacccaataa aagattatta tattgcatac cttttcttgc cataccattt
     7981 agccgatcaa ttgagatct
//
