LOCUS       sequence-217990-        7773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitous expression of one or multiple sgRNAs in Drosophila. 
            Selection marker: Mini-white..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  TITLE     crisprflydesign.org
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7773)
  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            "Unpublished"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: pCFD5_w
FEATURES             Location/Qualifiers
     source          1..7773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(65..83)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(104..120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(104..120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(117..139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    128..144
                     /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(152..158,159..176,177..182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    197..218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(335..352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(506..1094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(586..605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1265..2056,2057..2125))
                     /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     1888..1907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2126..2230)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2256..2711)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2393..2414)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2605..2624
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2838..2860
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2852..2869
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2853..2869
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2876..2895
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2876..2894
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2928..2946)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(3398..3469,3871..4144,4219..4873,4935..5250,5454..5585,
                     5656..6270)
                     /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"
     promoter        6493..6913
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        7019..7094
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        7181..7256
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    7550..7619
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gcgtcagcgg gttctcgacg gtcacggcgg gcatgtcgac actagttcta gccagctttt
       61 gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg
      121 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      181 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      241 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      301 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      361 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      421 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      541 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      601 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      661 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      721 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      781 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      841 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      901 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      961 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1021 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1081 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1141 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1201 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1261 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1321 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1381 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     1441 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1501 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1561 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1621 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1681 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1741 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1801 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1861 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1921 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1981 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2041 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2101 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2161 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2221 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     2281 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2341 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2401 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2461 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2521 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2581 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2641 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2701 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2761 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2821 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata
     2881 cgactcacta tagggcgaat tgggtacgta ccgggcccct agtatgtatg taagttaata
     2941 aaacccattt ttgcggaaag tagataaaaa aaacattttt tttttttact gcactggata
     3001 tcattgaact tatctgatca gttttaaatt tacttcgatc caagggtatt tgatgtacca
     3061 ggttctttcg attacctctc actcaaaatg acattccact caaagtcagc gctgtttgcc
     3121 tccttctctg tccacagaaa tatcgccgtc tctttcgccg ctgcgtccgc tatctctttc
     3181 gccaccgttt gtagcgttac gtagcgtcaa tgtccgcctt cagttgcatt ttgtcagcgg
     3241 tttcgtgacg aagctccaag cggtttacgc catcaattaa acacaaagtg ctgtgccaaa
     3301 actcctctcg cttcttattt ttgtttgttt tttgagtgat tggggtggtg attggttttg
     3361 ggtgggtaag caggggaaag tgtgaaaaat cccggcaatg ggccaagagg atcaggagct
     3421 attaattcgc ggaggcagca aacacccatc tgccgagcat ctgaacaatg tgagtagtac
     3481 atgtgcatac atcttaagtt cacttgatct ataggaactg cgattgcaac atcaaattgt
     3541 ctgcggcgtg agaactgcga cccacaaaaa tcccaaaccg caattgcaca aacaaatagt
     3601 gacacgaaac agattattct ggtagctgtt ctcgctatat aagacaattt ttgagatcat
     3661 atcatgatca agacatctaa aggcattcat tttcgactat attctttttt acaaaaaata
     3721 taacaaccag atattttaag ctgatcctag atgcacaaaa aataaataaa agtataaacc
     3781 tacttcgtag gatacttcgg ggtacttttt gttcggggtt agatgagcat aacgcttgta
     3841 gttgatattt gagatcccct atcattgcag ggtgacagcg gagcggcttc gcagagctgc
     3901 attaaccagg gcttcgggca ggccaaaaac tacggcacgc tccggccacc cagtccgccg
     3961 gaggactccg gttcagggag cggccaacta gccgagaacc tcacctatgc ctggcacaat
     4021 atggacatct ttggggcggt caatcagccg ggctccggat ggcggcagct ggtcaaccgg
     4081 acacgcggac tattctgcaa cgagcgacac ataccggcgc ccaggaaaca tttgctcaag
     4141 aacggtgagt ttctattcgc agtcggctga tctgtgtgaa atcttaataa agggtccaat
     4201 taccaatttg aaactcagtt tgcggcgtgg cctatccggg cgaacttttg gccgtgatgg
     4261 gcagttccgg tgccggaaag acgaccctgc tgaatgccct tgcctttcga tcgccgcagg
     4321 gcatccaagt atcgccatcc gggatgcgac tgctcaatgg ccaacctgtg gacgccaagg
     4381 agatgcaggc caggtgcgcc tatgtccagc aggatgacct ctttatcggc tccctaacgg
     4441 ccagggaaca cctgattttc caagccatgg tgcggatgcc acgacatctg acctatcggc
     4501 agcgagtggc ccgcgtggat caggtgatcc aggagctttc gctcagcaaa tgtcagcaca
     4561 cgatcatcgg tgtgcccggc agggtgaaag gtctgtccgg cggagaaagg aagcgtctgg
     4621 cattcgcctc cgaggctcta accgatccgc cgcttctgat ctgcgatgag cccacctccg
     4681 gactggactc ctttaccgcc cacagcgtcg tccaggtgct gaagaagctg tcgcagaagg
     4741 gcaagaccgt catcctgacc attcatcagc cgtcatccga gctgtttgag ctctttgaca
     4801 agatccttct gatggccgag ggcagggtag ctttcttggg cactcccagc gaagccgtcg
     4861 acttcttttc ctagtgagtt cgatgtgttt attaagggta tctagtatta cataacatct
     4921 caactcctat ccagcgtggg tgcccagtgt cctaccaact acaatccggc ggacttttac
     4981 gtacaggtgt tggccgttgt gcccggacgg gagatcgagt cccgtgatcg gatcgccaag
     5041 atatgcgaca attttgccat tagcaaagta gcccgggata tggagcagtt gttggccacc
     5101 aaaaatctgg agaagccact ggagcagccg gagaatgggt acacctacaa ggccacctgg
     5161 ttcatgcagt tccgggcggt cctgtggcga tcctggctgt cggtgctcaa ggaaccactc
     5221 ctcgtaaaag tgcgacttat tcagacaacg gtgagtggtt ccagtggaaa caaatgatat
     5281 aacgcttaca attcttggaa acaaattcgc tagattttag atagaattgc ctgattccac
     5341 acccttctta gtttttttca atgagatgta tagtttatag ttttgcagaa gataaataaa
     5401 tttcatttaa ctcgcgaata ttaatgagat gcgagtaaca ttttaatttg cagatggttg
     5461 ccatcttgat tggcctcatc tttttgggcc aacaactcac gcaagtgggt gtgatgaata
     5521 tcaacggagc catcttcctc ttcctgacca acatgacctt tcaaaacgtc tttgccacga
     5581 taaatgtaag tcatgtttag aatacatttg catttcaata atttactaac tttctaatga
     5641 atcgattcga tttaggtgtt cacctcagag ctgccagttt ttatgaggga ggcccgaagt
     5701 cgactttatc gctgtgacac atactttctg ggcaaaacga ttgccgaatt gccgcttttt
     5761 ctcacagtgc cactggtttt cacggcgatt gcctatccga tgatcggact gcgggccgga
     5821 gtgctgcact tcttcaactg cctggcgctg gtcactctgg tggccaatgt gtcaacgtcc
     5881 ttcggatatc taatatcctg cgccagctcc tcgacctcga tggcgctgtc tgtgggtccg
     5941 ccggttatca taccattcct gctctttggc ggcttcttct tgaactcggg ctcggtgcca
     6001 gtatacctca aatggttgtc gtacctctca tggttccgtt acgccaacga gggtctgctg
     6061 attaaccaat gggcggacgt ggagccgggc gaaattagct gcacatcgtc gaacaccacg
     6121 tgccccagtt cgggcaaggt catcctggag acgcttaact tctccgccgc cgatctgccg
     6181 ctggactacg tgggtctggc cattctcatc gtgagcttcc gggtgctcgc atatctggct
     6241 ctaagacttc gggcccgacg caaggagtag ccgacatata tccgaaataa ctgcttgttt
     6301 ttttttttac cattattacc atcgtgttta ctgtttattg ccccctcaaa aagctaatgt
     6361 aattatattt gtgccaataa aaacaagata tgacctatag aatacaagta tttccccttc
     6421 gaacatcccc acaagtagac tttggatttg tttctaacca aaagacttac acacctgcat
     6481 accttagaat tcttttttgc tcacctgtga ttgctcctac tcaaatacaa aaacatcaaa
     6541 ttttctgtca ataaagcata tttatttata tttattttac aggaaagaat tccttttaaa
     6601 gtgtatttta acctataatg aaaaacgatt aaaaaaaata cataaaataa ttcgaaaatt
     6661 tttgaatagc ccaggttgat aaaaattcat ttcatacgtt ttataactta tgcccctaag
     6721 tattttttga ccatagtgtt tcaattctac attaatttta cagagtagaa tgaaacgcca
     6781 cctactcagc caagaggcga aaaggttagc tcgccaagca gagagggcgc cagtgctcac
     6841 tactttttat aattctcaac ttctttttcc agactcagtt cgtatatata gacctatttt
     6901 caatttaacg tcggggcttt gagtgtgtgt agacatcaag catcggtggt tcagtggtag
     6961 aatgctcgcc tgccacgcgg gcggcccggg ttcgattccc ggccgatgca gggtcttcgt
     7021 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     7081 caccgagtcg gtgctaacaa agcaccagtg gtctagtggt agaatagtac cctgccacgg
     7141 tacagacccg ggttcgattc ccggctggtg cagaagacct gttttagagc tagaaatagc
     7201 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     7261 ttgcctacct ggagcctgag agttgttcaa taaaataaaa atgtttcgtt tttttgcttt
     7321 cgccagtatt tattattttt catcaatatg tattcaattt ggtatgtatt tagtaattgt
     7381 aatatataga caatggtttt ccgttgacgt acatacatct gacgtgtgtt tatttagaca
     7441 taatagttat gttttcacat ctttttaatg ttcgcttaat gcgtatgcat tctagagtgg
     7501 tgtgccaacc aacaacggat ccactagtgt cgacgatgta ggtcacagtc tcgaagccgc
     7561 ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc tcacccatct
     7621 ggtccatcat gatgaacggg tcgaggtggc ggtagttgat cccggcgaac gcgcggcgca
     7681 ccgggaagcc ctcgccctcg aaaccgctgg gcgcggtggt cacggtgagc acgggacgtg
     7741 cgacggcgtc ggcgggtgcg gatacgcggg gca
//
