LOCUS       PD0056_pIVTRup(original)-dCas9-VP64        8618 bp    DNA     circular UNA 30-JAN-2020
DEFINITION  Concatenation of 2 sequences.
ACCESSION   urn.local...6o-bsz22pg
VERSION     urn.local...6o-bsz22pg
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     promoter        75..93
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     primer_bind     145..205
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG
                     AGAGAAAAGAAGAG"
                     /Mismatches=0
                     /annotation_group="bcca1724-ec0c-4a59-88d9-c9a931922145"
                     /label="pIVTRup-BsmBI-FWD"
     primer_bind     145..173
                     /created_by="primer3"
                     /%GC="48.3"
                     /Tm="65.7"
                     /Hairpin_Tm="43.2"
                     /Self_Dimer_Tm="14.2"
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACGAC"
                     /annotation_group="2086465780022132c09f44-f0f0-4e55-86cd-c
                     8d4ea0a4196dCas9-VP64"
                     /label="145 F"
     primer_bind     145..171
                     /created_by="primer3"
                     /%GC="48.1"
                     /Tm="64.1"
                     /Hairpin_Tm="41.3"
                     /Self_Dimer_Tm="10.9"
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACG"
                     /annotation_group="20768612700143b944fd03-076d-4ff6-a986-e
                     13ea32485addCas9-VP64"
                     /label="145 F"
     primer_bind     145..171
                     /created_by="primer3"
                     /%GC="48.1"
                     /Tm="64.1"
                     /Hairpin_Tm="41.3"
                     /Self_Dimer_Tm="10.9"
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACG"
                     /annotation_group="20864657079419d81c3f89-83ec-4318-b4f4-e
                     629e41ca8bedCas9-VP64"
                     /label="145 F"
     misc_feature    145..205
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG
                     AGAGAAAAGAAGAG"
                     /created_by="User"
                     /label="pIVTRup_FWD"
                     /note="Geneious type: primer_bind_reverse"
     promoter        165..183
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     misc_feature    182..231
                     /Sequence="GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC
                     GAT"
                     /created_by="User"
                     /label="3'UTR"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    223..228
                     /Sequence="GCCACC"
                     /Hairpin_Tm="None"
                     /Tm="2.1"
                     /Self_Dimer_Tm="None"
                     /%GC="83.3"
                     /created_by="primer3"
                     /Color="#c3d9ff"
                     /modified_by="User"
                     /label="Kozak"
                     /note="Geneious type: primer_bind_reverse"
     source          <232..>4644
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /label="source synthetic DNA construct"
     misc_feature    232..4647
                     /Source="referenceToDocument: urn:local:.:11r-bsz22tr"
                     /label="PCR_dCas9-VP64 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    232..252
                     /Sequence="ATGAAAAGGCCGGCGGCCACG"
                     /Hairpin_Tm="68.2"
                     /Tm="68.7"
                     /Self_Dimer_Tm="56.1"
                     /%GC="66.7"
                     /created_by="primer3"
                     /label="mRNA_FWD"
                     /note="Geneious type: primer_bind_reverse"
     CDS             235..282
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /label="nucleoplasmin NLS"
     CDS             283..4383
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system, mutated to improve
                     targeting specificity (Slaymaker et al., 2016)"
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE
                     SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM
                     IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS
                     RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
                     LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL
                     KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL
                     NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
                     VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP
                     KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED
                     YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL
                     FEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL
                     KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV
                     KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH
                     PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL
                     TRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG
                     FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY
                     KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM
                     PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA
                     KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE
                     LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK
                     HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
                     AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /label="eSpCas9(1.1)"
     CDS             4438..4458
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             4483..4632
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDD
                     FDLDML"
                     /label="VP64"
     misc_feature    complement(4618..4644)
                     /Sequence="TGTACAGTTAATCAGCATGTCCAGGTC"
                     /Hairpin_Tm="37.4"
                     /Tm="62.9"
                     /Self_Dimer_Tm="None"
                     /%GC="44.4"
                     /created_by="primer3"
                     /Extension="TCA"
                     /label="mRNA_REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4618..4644)
                     /Mismatches=0
                     /Extension="TCA"
                     /created_by="primer3"
                     /annotation_group="83730026820499b4fd79-f997-46fa-980f-761
                     d86051e2f"
                     /%GC="44.4"
                     /Tm="62.9"
                     /Hairpin_Tm="37.4"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGTACAGTTAATCAGCATGTCCAGGTC"
                     /label="dCas9-VP64_REV"
                     /note="Geneious type: primer_bind_reverse"
     intron          4633..4644
                     /created_by="User"
                     /Color="#bbbbbb"
                     /modified_by="User"
                     /label="still_include"
     misc_feature    4648..231
                     /Source="referenceToDocument: urn:local:.:11q-bsz22tr"
                     /label="101362 - pIVTRup - EcoRV (concatenated sequence
                     1)"
                     /note="Geneious type: Concatenated sequence"
     CDS             4648..4754
                     /created_by="User"
                     /modified_by="User"
                     /label="5'UTR"
     misc_feature    complement(4726..4754)
                     /created_by="primer3"
                     /%GC="48.3"
                     /Tm="64.7"
                     /Hairpin_Tm="42.3"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGCTTTA"
                     /annotation_group="204583527922344c6530a9-460a-4148-8528-6
                     7dca669e1eedCas9-VP64"
                     /label="4,754 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4727..4754)
                     /created_by="primer3"
                     /%GC="50.0"
                     /Tm="65.0"
                     /Hairpin_Tm="42.3"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGCTTT"
                     /annotation_group="204299583698684448d012-0260-42a9-8ef4-a
                     35f9a0b7991dCas9-VP64"
                     /label="4,754 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4727..4754)
                     /created_by="primer3"
                     /%GC="50.0"
                     /Tm="65.0"
                     /Hairpin_Tm="42.3"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGCTTT"
                     /annotation_group="20458352263935cb2dcaa0-53d5-4cc4-b4b1-d
                     faa44a67367dCas9-VP64"
                     /label="4,754 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4728..4754)
                     /created_by="primer3"
                     /%GC="51.9"
                     /Tm="64.8"
                     /Hairpin_Tm="42.3"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGCTT"
                     /annotation_group="2045835185379673893082-f473-4793-89bb-3
                     87c9f7a6d43dCas9-VP64"
                     /label="4,754 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4729..4754)
                     /created_by="primer3"
                     /%GC="53.8"
                     /Tm="64.5"
                     /Hairpin_Tm="42.3"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGCT"
                     /annotation_group="20458350995627827e47b3-5dca-4b16-875c-b
                     9c411c9eeb8dCas9-VP64"
                     /label="4,754 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4730..4754)
                     /Sequence="CTAGACCCTCACTTCCTACTCAGGC"
                     /Hairpin_Tm="42.3"
                     /Tm="63.4"
                     /Self_Dimer_Tm="None"
                     /%GC="56.0"
                     /created_by="primer3"
                     /modified_by="User"
                     /label="TEST"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(4739..>4754)
                     /Sequence="TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
                     TTTTTTTTCTAGACCCTCACTTCC"
                     /Mismatches=0
                     /annotation_group="502e7721-7782-4d3b-9be9-92d4ba58a06e"
                     /label="pIVTRup-BsmBI-REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    <4755..>4809
                     /Source="referenceToDocument: urn:local:.:11q-bsz22tr"
                     /label="101362 - pIVTRup - EcoRV (concatenated sequence
                     1)"
                     /note="Geneious type: Concatenated sequence"
     3'UTR           4755..4809
                     /created_by="User"
                     /label="polyA(55)"
     misc_feature    <4755..>4809
                     /Source="referenceToDocument: urn:local:.:11s-bsz22tr"
                     /label="pIVTRup-linearized (concatenated sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    4809^4810
                     /Original_Bases="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAA"
                     /label="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAA"
                     /note="Geneious type: Editing History Deletion"
     protein_bind    4948..4964
                     /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)."
                     /label="lac operator"
     promoter        complement(4972..5002)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     misc_feature    complement(4989..5009)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14038071231149050e9745-290a-4fb6-b28d-c
                     a153b0a5499"
                     /%GC="47.6"
                     /Tm="57.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCCAGGCTTTACACTTTATGC"
                     /label="PCR_0122"
                     /note="Geneious type: primer_bind_reverse"
     protein_bind    5017..5038
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     misc_feature    complement(5151..5170)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1403776977450805f8ae7b-bd2e-4604-a10a-9
                     74da51416ba"
                     /%GC="60.0"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="9.1"
                     /Sequence="CGAGTCAGTGAGCGAGGAAG"
                     /label="PCR_0083"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(5151..5170)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14038699644695b383b0fa-c18b-494d-801f-1
                     37bf77a815a"
                     /%GC="60.0"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="9.1"
                     /Sequence="CGAGTCAGTGAGCGAGGAAG"
                     /label="PCR_0179"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(5254..5274)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14037989634263fba3cd39-fba6-4340-9ef4-5
                     f06acc6ffe0"
                     /%GC="52.4"
                     /Tm="60.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GCTCACATGTTCTTTCCTGCG"
                     /label="PCR_0101"
                     /note="Geneious type: primer_bind_reverse"
     rep_origin      complement(5326..5914)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     misc_feature    complement(5366..5385)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="140381554982221595ef36-0dd9-4636-802a-6
                     87564bd9d2d"
                     /%GC="60.0"
                     /Tm="60.7"
                     /Hairpin_Tm="36.7"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACCTCTGACTTGAGCGTCG"
                     /label="PCR_0143"
                     /note="Geneious type: primer_bind_reverse"
     CDS             complement(6085..6945)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     misc_feature    complement(6312..6339)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14037644287920c476db44-236e-42b9-8806-1
                     a6101184f7b"
                     /%GC="46.4"
                     /Tm="63.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCGG"
                     /label="PCR_0020"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(6312..6339)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14037725174218e16ff1aa-8573-482c-a5d7-2
                     3015618788e"
                     /%GC="46.4"
                     /Tm="63.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCGG"
                     /label="PCR_0068"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(6313..6339)
                     /Mismatches=0
                     /Extension="GGCGAAAGGGGGATGTGCTGCAAGGCGATT"
                     /created_by="primer3"
                     /annotation_group="14037898207417a993242f-2f80-4c4a-9cfe-a
                     3268c60d587"
                     /%GC="44.4"
                     /Tm="62.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCG"
                     /label="PCR_0097"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(6421..6441)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="14039242471087745b0b08-3dbc-48f0-80d0-7
                     354fb47f8a8"
                     /%GC="52.4"
                     /Tm="59.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GAAGCCATACCAAACGACGAG"
                     /label="qPCR_001"
                     /note="Geneious type: primer_bind_reverse"
     CDS             complement(6963..7757)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                      (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label="NeoR/KanR"
     rep_origin      complement(8101..8529)
                     /direction="LEFT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     misc_feature    complement(8596..8618)
                     /created_by="primer3"
                     /%GC="65.2"
                     /Tm="67.9"
                     /Hairpin_Tm="38.5"
                     /Self_Dimer_Tm="29.3"
                     /Sequence="CACATCCCCCTTTCGCCAGCTGG"
                     /annotation_group="203191415526312ee42101-56e9-4ef3-888d-0
                     fb3171f6ac6dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8596..8618)
                     /created_by="primer3"
                     /%GC="65.2"
                     /Tm="67.9"
                     /Hairpin_Tm="38.5"
                     /Self_Dimer_Tm="29.3"
                     /Sequence="CACATCCCCCTTTCGCCAGCTGG"
                     /annotation_group="20335435370447f303f7b2-7388-4f5e-86fa-b
                     16e258e4ef4dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8596..8618)
                     /created_by="primer3"
                     /%GC="65.2"
                     /Tm="67.9"
                     /Hairpin_Tm="38.5"
                     /Self_Dimer_Tm="29.3"
                     /Sequence="CACATCCCCCTTTCGCCAGCTGG"
                     /annotation_group="20387848985707be971265-078f-4062-bcf9-4
                     40ddf3027b2dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8596..8618)
                     /created_by="primer3"
                     /%GC="65.2"
                     /Tm="67.9"
                     /Hairpin_Tm="38.5"
                     /Self_Dimer_Tm="29.3"
                     /Sequence="CACATCCCCCTTTCGCCAGCTGG"
                     /annotation_group="20397001941088963d7e27-08f9-4e2f-b74c-6
                     11bedef3c9bdCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8597..8618)
                     /created_by="primer3"
                     /%GC="63.6"
                     /Tm="66.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="6.8"
                     /Sequence="CACATCCCCCTTTCGCCAGCTG"
                     /annotation_group="20319154230399cfe1d10f-5ab2-4d71-a338-1
                     4badf143a2edCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8597..8618)
                     /created_by="primer3"
                     /%GC="63.6"
                     /Tm="66.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="6.8"
                     /Sequence="CACATCCCCCTTTCGCCAGCTG"
                     /annotation_group="203354370829712eb1c189-bb10-47e1-9c2d-f
                     7a72bc1ca31dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8597..8618)
                     /created_by="primer3"
                     /%GC="63.6"
                     /Tm="66.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="6.8"
                     /Sequence="CACATCCCCCTTTCGCCAGCTG"
                     /annotation_group="20387851033169c699df99-bd74-4dd6-803d-6
                     00a7eeaa5eddCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8597..8618)
                     /created_by="primer3"
                     /%GC="63.6"
                     /Tm="66.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="6.8"
                     /Sequence="CACATCCCCCTTTCGCCAGCTG"
                     /annotation_group="203970042580033687e050-f7e3-4f57-98a9-3
                     82e0c995928dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8598..8618)
                     /created_by="primer3"
                     /%GC="61.9"
                     /Tm="65.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACATCCCCCTTTCGCCAGCT"
                     /annotation_group="203191529299672ae9bc88-21c7-4af6-9cb4-4
                     816a9e76203dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8598..8618)
                     /created_by="primer3"
                     /%GC="61.9"
                     /Tm="65.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACATCCCCCTTTCGCCAGCT"
                     /annotation_group="20335436271524235caecc-dc00-4cc7-9724-0
                     17050ca759edCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8598..8618)
                     /created_by="primer3"
                     /%GC="61.9"
                     /Tm="65.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACATCCCCCTTTCGCCAGCT"
                     /annotation_group="20387850229882c74f3dc8-5840-4316-9772-0
                     65fadf0b138dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8598..8618)
                     /created_by="primer3"
                     /%GC="61.9"
                     /Tm="65.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACATCCCCCTTTCGCCAGCT"
                     /annotation_group="203970036020682552aba9-4754-43ce-964b-f
                     f8949dad1a8dCas9-VP64"
                     /label="8,917 R"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(8611..13)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="140378105867366c6750c1-dd1c-4c58-baa7-5
                     cf65a2165dd"
                     /%GC="57.1"
                     /Tm="63.9"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="AATCGCCTTGCAGCACATCCC"
                     /label="PCR_0093"
                     /note="Geneious type: primer_bind_reverse"
ORIGIN      
        1 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
       61 cggccagtga attgtaatac gactcactat agggcgaatt gggccctcta gatgcatgct
      121 cgagcggccg ccagtgtgat ggatttggac cctcgtacag aagctaatac gactcactat
      181 agggaaataa gagagaaaag aagagtaaga agaaatataa gagccaccga tatgaaaagg
      241 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggacaagaa gtacagcatc
      301 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
      361 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
      421 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
      481 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
      541 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
      601 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
      661 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
      721 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
      781 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
      841 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
      901 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
      961 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
     1021 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     1081 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     1141 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     1201 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     1261 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     1321 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     1381 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     1441 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     1501 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     1561 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     1621 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     1681 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     1741 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     1801 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     1861 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     1921 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     1981 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     2041 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     2101 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     2161 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     2221 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     2281 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     2341 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     2401 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     2461 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     2521 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     2581 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     2641 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     2701 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     2761 ctggacatca accggctgtc cgactacgat gtggaccaca tcgtgcctca gagctttctg
     2821 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaaggcccg gggcaagagc
     2881 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     2941 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     3001 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     3061 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     3121 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     3181 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     3241 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     3301 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     3361 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     3421 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     3481 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     3541 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     3601 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     3661 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     3721 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     3781 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     3841 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     3901 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     3961 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     4021 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     4081 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     4141 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     4201 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     4261 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     4321 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     4381 gacagcgctg gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagcggacct
     4441 aagaaaaaga ggaaggtggc ggccgctgga tccggacggg ctgacgcatt ggacgatttt
     4501 gatctggata tgctgggaag tgacgccctc gatgattttg accttgacat gcttggttcg
     4561 gatgcccttg atgactttga cctcgacatg ctcggcagtg acgcccttga tgatttcgac
     4621 ctggacatgc tgattaactg tacatgaatc gctgccttct gcggggcttg ccttctggcc
     4681 atgcccttct tctctccctt gcacctgtac ctcttggtct ttgaataaag cctgagtagg
     4741 aagtgagggt ctagaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4801 aaaaaaaaac gatcaataac cgacactcct gcgtcgccta cgcgctacga tctgcagaat
     4861 tccagcacac tggcggccgt tactagtgga tccgagctcg gtaccaagct tggcgtaatc
     4921 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4981 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5041 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5101 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5161 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5221 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5281 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5341 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5401 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5461 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5521 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5581 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5641 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5701 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5761 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5821 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5881 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5941 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6001 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6061 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6121 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6181 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6241 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6301 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6361 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6421 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6481 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6541 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6601 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6661 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6721 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6781 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6841 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6901 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6961 attcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
     7021 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
     7081 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     7141 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     7201 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
     7261 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     7321 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     7381 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     7441 tgggatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     7501 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     7561 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     7621 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     7681 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     7741 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
     7801 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
     7861 accttaccag agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc
     7921 cagtctagct atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg
     7981 ttttcccttg tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc
     8041 ggactggctt tctacgtgtt ccgcttcctt tagcagccct tgcgccctga attttgttaa
     8101 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     8161 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     8221 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     8281 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     8341 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     8401 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     8461 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     8521 agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     8581 ctcttcgcta ttacgccagc tggcgaaagg gggatgtg
//
