LOCUS       Exported                8154 bp ds-DNA     circular SYN 05-JAN-2023
DEFINITION  Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9 and 
            U6>sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    pDCC6
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 Jan 5, 2023 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8154
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     regulatory      1099..1108
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1108..1173
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1180..1200
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1225..5325
                     /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             5329..5349
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(5366..5390)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          5482..5547
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5677..5697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6122..6256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6159..6178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6213..6232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             6353..7213
                     /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(6571..6590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7384..7972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7873..7892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8126..8143
                     /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 ctaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat
     1141 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     1201 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     1261 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     1321 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     1381 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     1441 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     1501 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     1561 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     1621 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     1681 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     1741 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     1801 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     1861 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     1921 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     1981 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     2041 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     2101 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     2161 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     2221 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     2281 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     2341 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     2401 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     2461 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     2521 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     2581 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     2641 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     2701 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     2761 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     2821 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     2881 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     2941 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     3001 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     3061 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     3121 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     3181 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     3241 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     3301 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     3361 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     3421 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     3481 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     3541 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     3601 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     3661 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     3721 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     3781 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     3841 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     3901 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     3961 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     4021 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     4081 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     4141 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     4201 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     4261 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     4321 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     4381 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     4441 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     4501 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     4561 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     4621 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     4681 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     4741 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     4801 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     4861 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     4921 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     4981 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     5041 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     5101 tacaacaagc accgggataa gcccatcaga gagcaggcca agaatatcat ccacctgttt
     5161 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     5221 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     5281 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacagccc caagaagaag
     5341 agaaaggtgg aggccagcta ataggaccca gctttcttgt acaaagtggt gacgtaagct
     5401 agcaggatct ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct
     5461 acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta
     5521 ctgattctaa ttgtttgtgt attttagatt ccaacctatg gaactgatga atgggagcag
     5581 tggtggaatg cctttaatga ggaaaacctg ttttgctcag aagaaatgcc atctagtgat
     5641 gatgaggcta ctgctgactc tcaacattct actcctccaa aaaagaagag aaaggtagtt
     5701 gaccccaagg actttccttc agaattgcta agttttttga gtcatgctgt gtttagtaat
     5761 agaactcttg cttgctttgc tatttacacc acaaaggaaa aagctgcact gctatacaag
     5821 aaaattatgg aaaaatattc tgtaaccttt ataagtaggc ataacagtta taatcataac
     5881 atactgtttt ttcttactcc acacaggcat agagtgtctg ctattaataa ctatgctcaa
     5941 aaattgtgta cctttagctt tttaatttgt aaaggggtta ataaggaata tttgatgtat
     6001 agtgccttga ctagagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     6061 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     6121 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6181 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6241 ttatcatgtc tggatcccgt ttaaactacg cgtaattcaa acagggttct ggcgtcgttc
     6301 tcgtactgtt ttccccaggc cagtgcttta gcgttattga aaaaggaaga gtatgagtat
     6361 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6421 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6481 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6541 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6601 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6661 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6721 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6781 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6841 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6901 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6961 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7021 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7081 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7141 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7201 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7261 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7321 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7381 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7441 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7861 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7921 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7981 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8041 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     8101 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaga
//
