LOCUS       sequence-381276-        8154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9 and 
            U6>sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8154)
  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 8154)
  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: pDCC6
FEATURES             Location/Qualifiers
     source          1..8154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..460
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        479..554
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      1076..1085
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1085..1150
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1157..1177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1202..5302
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAKNIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5306..5326
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(5343..5367)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5459..5524
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5654..5674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6099..6233
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6136..6155)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6190..6209
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(6330..6398,6399..7190)
                     /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(6548..6567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7361..7949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7850..7869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8103..8120
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca ggcaactcgt gaaaggtagg cggatcagcg
       61 gttcgacttg cagcctgaaa tacggcacga gtaggaaaag ccgagtcaaa tgccgaatgc
      121 agagtctcat tacagcacaa tcaactcaag aaaaactcga cactttttta ccatttgcac
      181 ttaaatcctt ttttattcgt tatgtatact ttttttggtc cctaaccaaa acaaaaccaa
      241 actctcttag tcgtgcctct atatttaaaa ctatcaattt attatagtca ataaatcgaa
      301 ctgtgttttc aacaaacgaa caataggaca ctttgattct aaaggaaatt ttgaaaatct
      361 taagcagagg gttcttaaga ccatttgcca attcttataa ttctcaactg ctctttcctg
      421 atgttgatca tttatatagg tatgttttcc tcaatacttc gggtcttcga gaagacctgt
      481 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      541 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      601 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaatccca aaacaaactg
      661 gttattgtgg taggtcattt gtttggcaga aagaaaactc gagaaatttc tctggccgtt
      721 attcgttatt ctctcttttc tttttgggtc tctccctctc tgcactaatg ctctctcact
      781 ctgtcacaca gtaaacggca tactgctctc gttggttcga gagagcgcgc ctcgaatgtt
      841 cgcgaaaaga gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt
      901 caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg
      961 aacaagctaa acaatctgca gccaagctcg ataagcttgt tcgaatctcg agtgcgcgct
     1021 tccggaggta tacacctagg cggtaccact gcagtgaatt cggagctcta ccggtgccac
     1081 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
     1141 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
     1201 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
     1261 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     1321 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
     1381 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     1441 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     1501 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     1561 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     1621 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     1681 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1741 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1801 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     1861 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     1921 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     1981 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     2041 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     2101 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     2161 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     2221 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     2281 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     2341 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     2401 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     2461 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     2521 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     2581 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     2641 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     2701 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2761 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     2821 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     2881 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     2941 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     3001 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     3061 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     3121 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     3181 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     3241 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     3301 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     3361 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     3421 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     3481 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     3541 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     3601 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     3661 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3721 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     3781 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     3841 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     3901 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     3961 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     4021 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     4081 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     4141 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     4201 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     4261 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     4321 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     4381 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     4441 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     4501 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     4561 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     4621 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     4681 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4741 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4801 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     4861 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     4921 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     4981 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     5041 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     5101 catcagagag caggccaaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     5161 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     5221 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     5281 cctgtctcag ctgggaggcg acagccccaa gaagaagaga aaggtggagg ccagctaata
     5341 ggacccagct ttcttgtaca aagtggtgac gtaagctagc aggatctttg tgaaggaacc
     5401 ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt
     5461 aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt
     5521 ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga
     5581 aaacctgttt tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgactctca
     5641 acattctact cctccaaaaa agaagagaaa ggtagttgac cccaaggact ttccttcaga
     5701 attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat
     5761 ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt
     5821 aacctttata agtaggcata acagttataa tcataacata ctgttttttc ttactccaca
     5881 caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt
     5941 aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa
     6001 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     6061 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     6121 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6181 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcccgttta
     6241 aactacgcgt aattcaaaca gggttctggc gtcgttctcg tactgttttc cccaggccag
     6301 tgctttagcg ttattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6361 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6421 taaaggatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6481 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6541 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6601 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6661 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6721 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6781 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6841 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6901 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6961 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7021 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7081 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7141 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7201 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7261 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7321 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7381 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7441 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7501 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7561 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7621 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7681 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7741 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7801 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7861 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7921 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7981 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     8041 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     8101 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctc
//
