LOCUS       pCMV-SpCas9-VRQR(R221K)-P2A-EGFP_(LRF95)        8452 bp    DNA     circular SYN 05-OCT-2025
DEFINITION  human codon optimized SpCas9 nuclease.
ACCESSION   urn.local...4f-jqra7jq
KEYWORDS    .
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     promoter        8218..129
                     /SerialCloner_Color="&h03C000"
                     /SerialCloner_Show="True"
                     /SerialCloner_Protect="True"
                     /SerialCloner_Arrow="True"
                     /X-Transferred_From="3.
                     pCAG-nls-Csy4-FokI-T2A-dCas9-recoded (C-terminal fusion) -
                     linker variants; 6.pCAG-nls-FokI-Csy4-T2A-Cas9-recoded
                     (N-terminal fusion) -linker variants; JDS246;
                     pCAG-Csy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64 recoded
                     (BPK376); pCAG-Csy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64
                     recoded (BPK376frameshift);
                     pCAG-Csy4-P2A-dCas9-NLS-FLAG-VP64 recoded;
                     pCAG-Csy4-T2A-Cas9-VP64 recoded (BPK1);
                     pCAG-Csy4-T2A-Cas9-VP64 recoded (BPK2a);
                     pCAG-Csy4-T2A-Cas9-recoded (SQT834);
                     pCAG-Csy4-T2A-FokI-dCas9-recoded-nls (JAF1484);
                     pCAG-Csy4-T2A-KRAB-dCas9-recoded-nlsFLAG (NW9);
                     pCAG-Csy4-T2A-NLS-FLAG-NLS-VP64-dCas9-recoded (making
                     p125); pCAG-Csy4-T2A-NLS-FLAG-NLS-dCas9-KRAB recoded
                     (BPK353); pCAG-Csy4-T2A-NLS-FLAG-NLS-dCas9-VP64 recoded
                     (BPK326); pCAG-Csy4-T2A-NLS-FLAG-NLS-dCas9-VP64 recoded NO
                     CTERM FLAG;
                     pCAG-Csy4-T2A-VP64-NLS/FLAG/NLS-dCas9-recoded-nlsFLAG
                     (BPK328); pCAG-Csy4-T2A-VP64-dCas9 linker variant BbsI
                     cassette; pCAG-Csy4-T2A-VP64-dCas9-recoded-nlsFLAG
                     (BPK285); pCAG-Csy4-T2A-VP64-dCas9-recoded-nlsFLAG
                     (BPK286); pCAG-Csy4-T2A-dCas9-KRAB recoded (NW8);
                     pCAG-Csy4-T2A-dCas9-VP64 recoded (BPK3);
                     pCAG-Csy4-T2A-dCas9-VP64 recoded NO FLAG (BPK373 or 374?);
                     pCAG-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64;
                     pCAG-NLS-FLAG-NLS-dCas9-VP64 recoded;
                     pCAG-NLS-FLAG-VP64-dCas9-recoded;
                     pCAG-NLS-VP64-dCas9-recoded; pCAG-NLS-dCas9-NLS-3xFLAG
                     recoded; pCAG-NLS-dCas9-VP64 recoded; pCAG-NLS-dCas9-VP64
                     recoded CopyXXXXX;
                     pCAG-NLS-hCsy4-P2A-NLS-FLAG-NLS-hdCas9-NLS-FLAG-VP64;
                     pCAG-NLS-hTtAgo (BPK633); pCAG-NLS-hTtAgo-NLS-3xFLAG
                     (BPK573); pCAG-NLS-hTtAgo-NLS-3xFLAG-VP64;
                     pCAG-NLS-hTtAgo-NLS-3xFLAG-VP64 D478A/D546A;
                     pCAG-NLS-hTtAgo-NLS-3xFLAG-VP64 D478N/D546N;
                     pCAG-VP64-NLS-FLAG-NLS-dCas9-recoded-NLS-FLAG (BPK329);
                     pCAG-VP64-NLS-hTtAgo; pCAG-VP64-dCas9-recod linker variant
                     BbsI cassette; pCAG-VP64-dCas9-recoded GGGGSx5 linker;
                     pCAG-VP64-dCas9-recoded-NLS-FLAG (BPK278);
                     pCAG-VP64-hCsy4-NLS-P2A-NLS-hdCas9-NLS-FLAG-VP64;
                     pCAG-dCas9-VP64;
                     pCAG-hCsy4-NLS-P2A-NLS-FLAG-NLS-hdCas9-NLS-FLAG-VP64
                     (BPK553);
                     pCAG-hCsy4-NLS-VP64-P2A-NLS-hdCas9-NLS-FLAG-VP64;
                     pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-3xFLAG-KRAB
                     (BPK550); pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64
                     delta175-307 (BPK544);
                     pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64 recoded;
                     pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64 recoded
                     (BPK447);
                     pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64-GG recoded
                     (BPK531); pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-KRAB
                     (BPK454); pCAG-hCsy4-P2A-NLS-FLAG-NLS-dCas9-NLS-VP64
                     recoded (BPK453); pCAG-hCsy4-P2A-NLS-dCas9-NLS-FLAG-VP64
                     recoded (BPK546); pCAG-hCsy4-P2A-NLS-dCas9-NLS-KRAB
                     (BPK548); pCAG-hCsy4-P2A-dCas9-NLS-FLAG-VP64 recoded
                     (BPK422); pCAG-hCsy4-P2A-dCas9-NLS-KRAB (BPK439);
                     pCAG-hCsy4-P2A-dCas9-NLS-VP64 recoded (BPK450);
                     pCAG-hCsy4-T2A-NLS-FLAG-NLS-dCas9-NLS-FLAG-VP64 recoded
                     (BPK428); pCAG-hCsy4-T2A-NLS-FLAG-NLS-dCas9-NLS-KRAB
                     (BPK444); pCAG-hCsy4-T2A-NLS-FLAG-NLS-dCas9-NLS-VP64
                     recoded (BPK437); pCAG-hCsy4-T2A-dCas9-NLS-FLAG-VP64
                     recoded (BPK426); pCAG-hCsy4-T2A-dCas9-NLS-KRAB (BPK442);
                     pCAG-hCsy4-T2A-dCas9-NLS-VP64 recoded (BPK435);
                     pCAG-hCsy4-T2A-nls-hFokI-dhCas9-nls-3XFLAG (SQT1601);
                     pCAG-hTtAgo; pCAG-hTtAgo-NLS; pCAG-hTtAgo-NLS-3xFLAG;
                     pCAG-nls-FokI-Csy4-T2A-dCas9-recoded (N-terminal fusion
                     CK1891); pCMV-NLS-hTtAgo-NLS-3xFLAG; pLenti-CAG-Puro-DEST;
                     pMLM3705; pMLM3705 annotation mess"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pCMV-T7-Cas9 (MLM3613), pCMV-T7-Cas9
                     delta NcoI"
                     /Transferred_Similarity="100.00%"
                     /modified_by="KleinstiverLab"
                     /label="CMV Enhancer"
     promoter        287..356
                     /label="CMV_promoter"
     promoter        377..396
                     /X-Transferred_From="pCRII-TOPO"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pCMV-T7-Cas9 (MLM3613), pCMV-T7-Cas9
                     delta NcoI"
                     /Transferred_Similarity="100.00%"
                     /label="T7 prom"
     motif           400..412
                     /created_by="User"
                     /label="Kozak"
     CDS             409..4512
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /Original_Translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
                     NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
                     DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
                     LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA
                     ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK
                     DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD
                     EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
                     GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
                     ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
                     NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK
                     VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL
                     EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ
                     SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA
                     IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
                     LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK
                     DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG
                     GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS
                     DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
                     AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF
                     ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT
                     VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII
                     KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
                     KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL
                     FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /modified_by="ben"
                     /label="SpCas9-VRQR+R221K"
     source          <412..>4512
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_difference 1069..1071
                     /created_by="ben"
                     /label="R221K"
     misc_feature    1069..1070
                     /Original_Bases="CG"
                     /label="CG"
                     /note="Geneious type: Editing History Replacement"
     misc_difference 3811..3813
                     /created_by="ben"
                     /label="D1135V"
     misc_feature    3812..3813
                     /Original_Bases="AC"
                     /label="AC"
                     /note="Geneious type: Editing History Replacement"
     misc_difference 4060..4062
                     /created_by="ben"
                     /modified_by="ben"
                     /label="G1218R"
     misc_feature    4060
                     /Original_Bases="G"
                     /label="G"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    4062
                     /Original_Bases="C"
                     /label="C"
                     /note="Geneious type: Editing History Replacement"
     misc_difference 4411..4413
                     /created_by="ben"
                     /label="R1335Q"
     misc_feature    4411..4412
                     /Original_Bases="AG"
                     /label="AG"
                     /note="Geneious type: Editing History Replacement"
     misc_difference 4417..4419
                     /created_by="ben"
                     /label="T1337R"
     misc_feature    4418..4419
                     /Original_Bases="CC"
                     /label="CC"
                     /note="Geneious type: Editing History Replacement"
     CDS             4513..4536
                     /created_by="Kleinstiver Lab 1"
                     /Color="#ff7400"
                     /modified_by="KleinstiverLab"
                     /label="linker"
     CDS             4537..4587
                     /created_by="Kleinstiver Lab 1"
                     /Color="#6bba70"
                     /modified_by="benjaminkleinstiver"
                     /label="BPNLS(SV40)"
     CDS             4588..4599
                     /created_by="Kleinstiver Lab 1"
                     /Color="#ff7400"
                     /modified_by="KleinstiverLab"
                     /label="linker"
     CDS             4600..4665
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVS
                     SDYKDHDGDYKDHDIDYKDDDDKAAG*"
                     /Color="#ffbbff"
                     /modified_by="ben"
                     /label="3xFLAG"
     CDS             4666..4674
                     /created_by="User"
                     /Color="#ff7400"
                     /modified_by="Kleinstiver Lab 1"
                     /label="linker"
     CDS             4675..4740
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAG
                     QAKKKKDYKDDDDKGSGATNFSLLKQAGDVEENPGPTEYKPTVRLATRDDVPRAVRTL
                     AAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPES
                     VEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGL
                     GSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKP
                     GA*"
                     /Color="#7700ee"
                     /modified_by="benjaminkleinstiver"
                     /label="P2A"
     source          <4681..>4740
                     /organism="lentiCRISPR v2"
                     /mol_type="other DNA"
     CDS             4741..5457
                     /created_by="User"
                     /Color="#008c00"
                     /modified_by="benjaminkleinstiver"
                     /label="EGFP"
     CDS             5458..5460
                     /Color="#000000"
                     /created_by="benjaminkleinstiver"
                     /label
     terminator      5509..5736
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVS
                     SDYKDHDGDYKDHDIDYKDDDDKAAG*"
                     /label="bGH_PA_terminator"
     misc_feature    complement(6194..6813)
                     /Imported_from="<a
                     href=""http://wishart.biology.ualberta.ca/PlasMapper"">Pla
                     sMapper</a>"
                     /X-Transferred_From="pBR322 origin"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pCAG-hCas9-nls-3XFLAG (SQT817),
                     pCAG-hCas9-nls-3XFLAG delta NcoI (BPK742), pCMV-T7-Cas9
                     (MLM3613), pCMV-T7-Cas9 delta NcoI"
                     /Transferred_Similarity="100.00%"
                     /label="pBR322 origin"
                     /note="Geneious type: origin of replication"
     CDS             complement(6968..7828)
                     /created_by="Kleinstiver Lab 4"
                     /Color="#c3d9ff"
                     /modified_by="KleinstiverLab"
                     /label="bla CDS"
     misc_feature    complement(7829..7927)
                     /invitrogen="""2330000"
                     /Transferred_From="pDEST15"
                     /Transferred_Similarity="100.00%"
                     /label="bla promoter"
                     /note="Geneious type: promoter prokaryotic"
     misc_feature    8452
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
ORIGIN      
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggacaagaag
      421 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
      481 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
      541 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
      601 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
      661 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
      721 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
      781 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
      841 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
      901 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
      961 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1021 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagaaa gctggaaaat
     1081 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     1141 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     1201 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     1261 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     1321 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     1381 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     1441 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     1501 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     1561 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     1621 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     1681 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     1741 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     1801 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     1861 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     1921 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     1981 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     2041 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     2101 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     2161 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     2221 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     2281 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     2341 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     2401 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     2461 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     2521 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     2581 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     2641 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     2701 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     2761 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     2821 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     2881 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     2941 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3001 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     3061 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     3121 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     3181 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     3241 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     3301 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     3361 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     3421 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     3481 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     3541 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     3601 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     3661 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     3721 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     3781 gactgggacc ctaagaagta cggcggcttc gtgagcccca ccgtggccta ttctgtgctg
     3841 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     3901 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     3961 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4021 gagctggaaa acggccggaa gagaatgctg gcctctgcca gagaactgca gaagggaaac
     4081 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     4141 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     4201 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     4261 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     4321 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     4381 aagtactttg acaccaccat cgaccggaag cagtacagaa gcaccaaaga ggtgctggac
     4441 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     4501 ctgggaggcg acggatccgg cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa
     4561 ttcgagccca agaagaagag gaaagtctcg agcggaggcg actacaaaga ccatgacggt
     4621 gattataaag atcatgacat cgattacaag gatgacgatg acaagggagg cggtggaagc
     4681 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     4741 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     4921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     5041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     5101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     5161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     5221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     5281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     5341 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     5401 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga
     5461 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5521 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5581 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5641 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5701 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5761 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5821 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5881 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5941 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6001 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6061 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6121 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6181 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6241 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6301 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6361 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     6421 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6481 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6541 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6601 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6661 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6721 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7921 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7981 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     8041 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8101 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     8161 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8221 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8281 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8341 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8401 gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat ca
//
