LOCUS       sequence-95389-e        8120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9-D10A
            nickase and U6>sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8120)
  AUTHORS   Gokcezade J, Sienski G, Duchek P
  TITLE     Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome 
            Editing in Drosophila.
  JOURNAL   G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 
            10.1534/g3.114.014126.
  PUBMED    25236734
REFERENCE   2  (bases 1 to 8120)
  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: "G3 
            (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 
            10.1534/g3.114.014126."
COMMENT     Sequence Label: pDCC5
FEATURES             Location/Qualifiers
     source          1..8120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        84..483
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        502..577
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1101..1125
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1131..1157
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1161..1181
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1191..5291
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAKNIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5295..5315
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(5332..5356)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5448..5513
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5643..5663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6088..6222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6125..6144)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6179..6198
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6319..6387,6388..7179)
                     /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     complement(6537..6556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7350..7938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7839..7858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8092..8109
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaggcaact
       61 cgtgaaaggt aggcggatca gcggttcgac ttgcagcctg aaatacggca cgagtaggaa
      121 aagccgagtc aaatgccgaa tgcagagtct cattacagca caatcaactc aagaaaaact
      181 cgacactttt ttaccatttg cacttaaatc cttttttatt cgttatgtat actttttttg
      241 gtccctaacc aaaacaaaac caaactctct tagtcgtgcc tctatattta aaactatcaa
      301 tttattatag tcaataaatc gaactgtgtt ttcaacaaac gaacaatagg acactttgat
      361 tctaaaggaa attttgaaaa tcttaagcag agggttctta agaccatttg ccaattctta
      421 taattctcaa ctgctctttc ctgatgttga tcatttatat aggtatgttt tcctcaatac
      481 ttcgggtctt cgagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt
      541 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat
      601 agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg
      661 ctctagaatc ccaaaacaaa ctggttattg tggtaggtca tttgtttggc agaaagaaaa
      721 ctcgagaaat ttctctggcc gttattcgtt attctctctt ttctttttgg gtctctccct
      781 ctctgcacta atgctctctc actctgtcac acagtaaacg gcatactgct ctcgttggtt
      841 cgagagagcg cgcctcgaat gttcgcgaaa agagcgccgg agtataaata gaggcgcttc
      901 gtctacggag cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct
      961 aagcaaataa acaagcgcag ctgaacaagc taaacaatct gcagccaagc tcgataagct
     1021 tgttcgaatc tcgagtgcgc gcttccggag gtatacacct aggcggtacc actgcagtga
     1081 attcggagct ctaccggtat acaagtttgt acaaaaaagc aggctcaatg tacccatacg
     1141 atgttccaga ttacgcttcg ccgaagaaaa agcgcaaggt cgaagcgtcc gacaagaagt
     1201 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     1261 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     1321 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     1381 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     1441 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     1501 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     1561 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     1621 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     1681 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     1741 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     1801 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     1861 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
     1921 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     1981 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     2041 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     2101 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     2161 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     2221 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     2281 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     2341 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     2401 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     2461 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     2521 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     2581 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     2641 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     2701 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     2761 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     2821 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     2881 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     2941 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     3001 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     3061 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     3121 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     3181 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     3241 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     3301 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     3361 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3421 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3481 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3541 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3601 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3661 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     3721 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     3781 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     3841 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     3901 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     3961 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     4021 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     4081 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4141 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4201 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4261 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4321 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4381 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4441 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4501 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4561 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     4621 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4681 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4741 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     4801 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     4861 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     4921 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     4981 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     5041 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     5101 aggccaagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5161 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     5221 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5281 tgggaggcga cagccccaag aagaagagaa aggtggaggc cagctaatag gacccagctt
     5341 tcttgtacaa agtggtgacg taagctagca ggatctttgt gaaggaacct tacttctgtg
     5401 gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat
     5461 ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattccaa
     5521 cctatggaac tgatgaatgg gagcagtggt ggaatgcctt taatgaggaa aacctgtttt
     5581 gctcagaaga aatgccatct agtgatgatg aggctactgc tgactctcaa cattctactc
     5641 ctccaaaaaa gaagagaaag gtagttgacc ccaaggactt tccttcagaa ttgctaagtt
     5701 ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa
     5761 aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa atattctgta acctttataa
     5821 gtaggcataa cagttataat cataacatac tgttttttct tactccacac aggcatagag
     5881 tgtctgctat taataactat gctcaaaaat tgtgtacctt tagcttttta atttgtaaag
     5941 gggttaataa ggaatatttg atgtatagtg ccttgactag agatcataat cagccatacc
     6001 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     6061 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     6121 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     6181 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcccgtttaa actacgcgta
     6241 attcaaacag ggttctggcg tcgttctcgt actgttttcc ccaggccagt gctttagcgt
     6301 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6361 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6421 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6481 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6541 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6601 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6661 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6721 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6781 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6841 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6901 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6961 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7021 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7081 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7141 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7201 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7981 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     8041 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     8101 gtgagcgagg aagcggaaga
//
