LOCUS       Exported                8577 bp ds-DNA     circular SYN 06-JUL-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8577)
  AUTHORS   Nishida
  TITLE     Direct Submission
  JOURNAL   Exported Jul 6, 2022 from SnapGene 4.2.11
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8577
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     primer_bind     complement(1..42)
                     /label=kn1303
                     /note="Hs-pCMV_start_R"
                     /note="color: ?; sequence: 
                     CCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTC"
     primer_bind     complement(57..76)
                     /label=kn1312
                     /note="phCMV100_R"
                     /note="color: ?; sequence: CGGAACTCCATATATGGGCT"
     primer_bind     complement(83..106)
                     /label=sy30
                     /note="Rv1 1906EUGI in"
                     /note="color: ?; sequence: GGCGGGCCATTTACCGTAAGTTAT"
     primer_bind     83..106
                     /label=sy31
                     /note="Fw2 1906EUGI in"
                     /note="color: ?; sequence: ATAACTTACGGTAAATGGCCCGCC"
     primer_bind     181..206
                     /label=hm436
                     /note="CMV pro mid top for 800-bp ANA sub,26"
                     /note="color: ?; sequence: CTTTCCATTGACGTCAATGGGTGGAG"
     primer_bind     228..245
                     /label=hm941
                     /note="CMVpro upstream of NdeI top,18"
                     /note="color: ?; sequence: TTGGCAGTACATCAAGTG"
     primer_bind     283..299
                     /label=sy31
                     /note="Fw2 1906EUGI in"
                     /note="color: ?; sequence: ATAACTTACGGTAAATGGCCCGCC"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     complement(389..419)
                     /label=hm356
                     /note="sy5 Nco-frag2 to remove SapI btm,31"
                     /note="color: ?; sequence: CTATCCACGCCCATTGATGTACTGCCAAAAC"
     primer_bind     481..499
                     /label=kn1304
                     /note="pCMVend100_F"
                     /note="color: ?; sequence: GGCACCAAAATCAACGGGA"
     primer_bind     512..532
                     /label=Sy496
                     /note="color: ?; sequence: 
                     TCTTTCCCTACACGACGCTCTTCCGATCTTCGTAACAACTCCGCCCCATT"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
                     /note="color: ?; sequence: CGCAAATGGGCGGTAGGCGTG"
     primer_bind     complement(539..557)
                     /label=Sy795
                     /note="color: ?; sequence: tacacgcctaccgcccatt"
     primer_bind     539..557
                     /label=Sy573
                     /note="color: ?; sequence: AATGGGCGGTAGGCGTGTA"
     primer_bind     551..571
                     /label=Sy575
                     /note="mEGFP 2nd"
                     /note="color: ?; sequence: 
                     TCTTTCCCTACACGACGCTCTTCCGATCTGCGTGTACGGTGGGAGGTCTA"
     primer_bind     559..583
                     /label=hm434
                     /note="CMV pro 3' top,25"
                     /note="color: ?; sequence: GGTGGGAGGTCTATATAAGCAGAGC"
     primer_bind     585..607
                     /label=sy100
                     /note="SY1-3 Fw EF1at¯O¦,58"
                     /note="color: ?; sequence: GGTTTAGTGAACCGTCAGATCCG"
     primer_bind     586..609
                     /label=sy104
                     /note="NiRFP Fw +overlap,59"
                     /note="color: ?; sequence: 
                     aatttcgacccggatccgtTTAGTGAACCGTCAGATCCGCT"
     primer_bind     587..609
                     /label=sy33
                     /note="NiRFP Fw +/-eEoverlap^X,58,Ef1a½c¶"
                     /note="color: ?; sequence: 
                     tcgtgaacaccgccaccatTTAGTGAACCGTCAGATCCGCT"
     primer_bind     complement(591..611)
                     /label=Sy469
                     /note="110 bp center Rv"
                     /note="color: ?; sequence: 
                     CCAGGATGGGCACCACCCCGGTGAACAGCtCCTCGCCCTTGCTCACCAtctacattcca
                     actgggccGGTAGATCTGAGTCCGGTAGCGCTAGCGGATCTGACGGTTCAC"
     primer_bind     591..609
                     /label=sy323
                     /note="mEGFP"
                     /note="color: ?; sequence: gtaGGTACCGTGAACCGTCAGATCCGCT"
     primer_bind     complement(593..610)
                     /label=Sy480
                     /note="color: ?; sequence: TAGCGGATCTGACGGTTC"
     primer_bind     complement(596..611)
                     /label=Sy466
                     /note="70 bp ¶E¸c·@15 Rv"
                     /note="color: ?; sequence: 
                     CCCTTGCTCACCAtctacattccaactgggccGGTAGATCTGAGTCCGGTAGCGCTAGC
                     GGATCTGACGG"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
                     /note="color: ?; sequence: TAATACGACTCACTATAGGG"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     626..644
                     /label=hm817
                     /note="5'-FAM T7 promoter,19"
                     /note="color: ?; sequence: TAATACGACTCACTATAGG"
     primer_bind     626..643
                     /label=hm444
                     /note="T7 promoter,18"
                     /note="color: ?; sequence: TAATACGACTCACTATAG"
     primer_bind     655..683
                     /label=hm1109
                     /note="bpNLS top 1 for GA w/ GAL110pro,44"
                     /note="color: ?; sequence: 
                     cgtcaaggagaaaaaaccatgaaacggacagccgacggaagcga"
     primer_bind     661..680
                     /label=hm1156
                     /note="NcoI-bipartite NLS,44"
                     /note="color: ?; sequence: 
                     cgtcaaggagaaaaaaccatggcaaaacggacagccgacggaag"
     primer_bind     complement(664..707)
                     /label=hm1110
                     /note="bpNLS btm 1 (inner),44"
                     /note="color: ?; sequence: 
                     cgcttcttctttggtgactcgaactcgcttccgtcggctgtccg"
     primer_bind     complement(690..729)
                     /label=al122
                     /note="color: ?; sequence: 
                     gctgtacttcttgtcgactttccgcttcttctttggtgac"
     primer_bind     complement(690..714)
                     /label=al130
                     /note="pAL036BE4Max,42"
                     /note="color: ?; sequence: 
                     ACGTAGCAGCGGTGTGAgactttccgcttcttctttggtgac"
     CDS             694..714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     primer_bind     700..734
                     /label=al123
                     /note="color: ?; sequence: 
                     aagaagcggaaagtcgacaagaagtacagcatcgg"
     primer_bind     714..734
                     /label=al131
                     /note="pAL036BE4Max,37"
                     /note="color: ?; sequence: 
                     atgtcGGcGGAGGcTCcgacaagaagtacagcatcgg"
     CDS             715..3873
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /note="This feature has 3 segments:
                        1:  715 ..  738 / #993366
                        2:  739 ..  741 / #cc99b2
                        3:  742 .. 3873 / #993366"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLAN"
     primer_bind     715..734
                     /label=hm1131
                     /note="human op Cas9 top1,20"
                     /note="color: ?; sequence: gacaagaagtacagcatcgg"
     primer_bind     726..755
                     /label=kn1051
                     /note="hCas9(D10A)_F"
                     /note="color: ?; sequence: cagcatcggcctggTcatcggcaccaactc"
     primer_bind     complement(991..1009)
                     /label=kn1056
                     /note="hCas9_500_R"
                     /note="color: ?; sequence: tgtggaagaagctgtcgtc"
     primer_bind     complement(1065..1084)
                     /label=hm1138
                     /note="human op Cas9 btm1,20"
                     /note="color: ?; sequence: cgtccacgatgttgccgaag"
     primer_bind     1334..1353
                     /label=hm1132
                     /note="human op Cas9 top2,20"
                     /note="color: ?; sequence: ccaaggccatcctgtctgcc"
     primer_bind     1462..1486
                     /label=Sy760
                     /note="color: ?; sequence: aacttcaagagcaacttcgacctgg"
     primer_bind     complement(1497..1515)
                     /label=kn1057
                     /note="hCas9_1k_R"
                     /note="color: ?; sequence: cttgctcagctgcagtttg"
     primer_bind     1915..1934
                     /label=hm1133
                     /note="human op Cas9 top3,20"
                     /note="color: ?; sequence: cagcggaccttcgacaacgg"
     primer_bind     complement(2001..2019)
                     /label=kn1058
                     /note="hCas9_1.5k_R"
                     /note="color: ?; sequence: gttgtccttcaggaatggg"
     primer_bind     2245..2266
                     /label=Sy761
                     /note="color: ?; sequence: agcctgctgtacgagtacttca"
     primer_bind     complement(2514..2532)
                     /label=kn1059
                     /note="hCas9_2k_R"
                     /note="color: ?; sequence: caggaagtccttgtccttg"
     primer_bind     2522..2541
                     /label=hm1134
                     /note="human op Cas9 top4,20"
                     /note="color: ?; sequence: aggacttcctggacaatgag"
     primer_bind     2697..2707
                     /label=kn2488
                     /note="tracr-in-UV5_R"
                     /note="color: ?; sequence: 
                     AACTTGCTATGCTGTTTTGAATGGTTccAACAAGATCATTATACGAGCCGGAAGC"
     primer_bind     complement(3036..3053)
                     /label=kn1060
                     /note="hCas9_2.5k_R"
                     /note="color: ?; sequence: attctctcgcggctgttc"
     primer_bind     complement(3065..3084)
                     /label=Sy797
                     /note="color: ?; sequence: cagctctttgatgccctctt"
     primer_bind     3067..3084
                     /label=Sy762
                     /note="color: ?; sequence: gagggcatcaaagagctg"
     primer_bind     3067..3084
                     /label=kn2157
                     /note="OsCas9_2300_F"
                     /note="color: ?; sequence: GAGGGCATCAAAGAGCTG"
     primer_bind     3137..3156
                     /label=hm1135
                     /note="human op Cas9 top5,20"
                     /note="color: ?; sequence: agaagctgtacctgtactac"
     primer_bind     complement(3213..3247)
                     /label=kn1054
                     /note="hCas9(H840A)_R"
                     /note="color: ?; sequence: 
                     agctctgaggcacgatagcgtccacatcgtagtcg"
     primer_bind     3213..3247
                     /label=kn1053
                     /note="hCas9(H840A)_F"
                     /note="color: ?; sequence: 
                     cgactacgatgtggacGCtatcgtgcctcagagct"
     primer_bind     complement(3552..3569)
                     /label=kn1061
                     /note="hCas9_3k_R"
                     /note="color: ?; sequence: acttcccggatcagcttg"
     primer_bind     3748..3767
                     /label=hm1136
                     /note="human op Cas9 top6,20"
                     /note="color: ?; sequence: tacaaggtgtacgacgtgcg"
     primer_bind     complement(3849..3872)
                     /label=al085
                     /note="V47,25"
                     /note="color: ?; sequence: ATTGGCCAGTGTAATCTCGGTCTTG"
     primer_bind     complement(3854..3898)
                     /label=al124
                     /note="color: ?; sequence: 
                     CAAACAGCACGTAGCAGCGGTGTGAgttggccagggtaatctcgg"
     primer_bind     3873..3893
                     /label=Sy741
                     /note="AN16_X-Ten_F"
                     /note="color: ?; sequence: 
                     gcaacacctgaaagcTCACACCGCTGCTACGTGCT"
     primer_bind     3873..3893
                     /label=Sy731
                     /note="color: ?; sequence: 
                     attatcaagaaaggcTCACACCGCTGCTACGTGCT"
     primer_bind     3873..3893
                     /label=Sy712
                     /note="Asuya0619"
                     /note="color: ?; sequence: 
                     attatcaagaaaggcTCACACCGCTGCTACGTGCT"
     primer_bind     3873..3892
                     /label=al161
                     /note="pAL058 (pAL052 PAPAPAP-Nter linker),40"
                     /note="color: ?; sequence: 
                     cccgctcctgcccccgctcccTCACACCGCTGCTACGTGC"
     CDS             3874..4236
                     /codon_start=1
                     /label=HsPmCDA1(30?150_W122E_W139Q)
                     /note="color: #00ccff"
                     /translation="SHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRK
                     VEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIEACKLYYEKNAR
                     NQIGLQNLRDNGVGLNV"
     primer_bind     3874..3893
                     /label=Sy626
                     /note="color: ?; sequence: 
                     GACGATAAATCAGGGTCACACCGCTGCTACGTGCT"
     primer_bind     3874..3893
                     /label=Sy623
                     /note="color: ?; sequence: 
                     GGAGTTCCTGCGGCATCACACCGCTGCTACGTGCT"
     primer_bind     3874..3892
                     /label=al107
                     /note="color: ?; sequence: TCACACCGCTGCTACGTGC"
     primer_bind     complement(3931..3952)
                     /label=hm087
                     /note="PmCDA1 btm seq,22"
                     /note="color: ?; sequence: TGTTGACGGCATAGCCCCAAAA"
     primer_bind     complement(3969..4008)
                     /label=hm932
                     /note="PmCDA1 dead mut E68A btm,40"
                     /note="color: ?; sequence: 
                     CTTTCGGATACTGAATATCgCAGCGTGTATTCCTCTCTCG"
     primer_bind     3990..4008
                     /label=hm933
                     /note="PmCDA1 dead mut E68A top,21"
                     /note="color: ?; sequence: GcGATATTCAGTATCCGAAAG"
     primer_bind     4010..4034
                     /label=Sy774
                     /note="color: ?; sequence: TGGAAGAGTATCTTCGGGATAATCC"
     primer_bind     complement(4125..4144)
                     /label=Sy668
                     /note="color: ?; sequence: TCAATGTGTGCCCGTTTCCC"
     primer_bind     complement(4130..4236)
                     /label=Sy667
                     /note="color: ?; sequence: 
                     CACGTTCAGGCCTACACCATTGTCCCTCAGATTtctGAGTCCTATCTGGTTCCGGGCAT
                     TCTTCTCGTAGTACAGCTTGCAGGCttcGATTTTCAATGTGTGCCCGT"
     primer_bind     4130..4236
                     /label=Sy666
                     /note="color: ?; sequence: 
                     ACGGGCACACATTGAAAATCgaaGCCTGCAAGCTGTACTACGAGAAGAATGCCCGGAAC
                     CAGATAGGACTCagaAATCTGAGGGACAATGGTGTAGGCCTGAACGTG"
     misc_feature    4150..4152
                     /label=W122E
                     /note="color: #a6acb3"
     primer_bind     complement(4182..4236)
                     /label=al108
                     /note="color: ?; sequence: 
                     CACGTTCAGGCCTACACCATTGTCCCTCAGATTctgGAGTCCTATCTGGTTCCGG"
     primer_bind     complement(4215..4236)
                     /label=al158
                     /note="pAL057 (pAL052 PAPAPAP-Cter linker),50"
                     /note="color: ?; sequence: 
                     tgcggccgggagcgggggcaggagcgggCACGTTCAGGCCTACACCATTG"
     primer_bind     complement(4217..4236)
                     /label=Sy738
                     /note="AN155_X-Ten_R"
                     /note="color: ?; sequence: 
                     tgtctcagagccagaCACGTTCAGGCCTACACCAT"
     primer_bind     complement(4217..4236)
                     /label=Sy624
                     /note="color: ?; sequence: CACGTTCAGGCCTACACCAT"
     unsure          4237..4269
                     /label=6xGS Linker
                     /note="color: #ccffcc"
     primer_bind     4245..4291
                     /label=al126
                     /note="color: ?; sequence: 
                     tggcagtgatgtcGGcGGAGGcTCcggcgagatccggaagcggcctc"
     primer_bind     complement(4248..4269)
                     /label=al125
                     /note="color: ?; sequence: gGAgCCTCCgCCgacatcactg"
     primer_bind     4250..4289
                     /label=hm1101
                     /note="Cas9(BE4max) middle top for split-047,40"
                     /note="color: ?; sequence: 
                     gtgatgtcGGcGGAGGcTCcggcgagatccggaagcggcc"
     CDS             4270..5211
                     /codon_start=2
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /note="color: #993366"
                     /translation="ARSGSGL*SRQTAKPGRSCGIRAGILPPCGKC*ACPK*IS*KRPR
                     CRQAASAKSLSCPRGTAIS*SPERRTGTLRSTAASTAPPWPILCWWWPKWKRASPRN*R
                     V*KSCWGSPSWKEAASRRIPSTFWKPRATKK*KRT*SSSCLSTPCSSWKTAGRECWPLP
                     ANCRRETNWPCPPNM*TSCTWPATMRS*RAPPRIMSRNSCLWNSTSTTWTRSSSRSASS
                     PRE*SWPTLIWTKCCPPTTSTGISPSESRPRISSTCLP*PIWEPLPPSSTLTPPSTGRG
                     TPAPKRCWTPP*STRASPACTRHGSTCLSWEV"
     primer_bind     4330..4351
                     /label=Sy763
                     /note="color: ?; sequence: gataagggccgggattttgcca"
     primer_bind     complement(4469..4487)
                     /label=kn1062
                     /note="hCas9_3.5k_R"
                     /note="color: ?; sequence: tcccagtccttctttctgg"
     primer_bind     4728..4747
                     /label=hm1137
                     /note="human op Cas9 top7,20"
                     /note="color: ?; sequence: aaacggccggaagagaatgc"
     primer_bind     complement(4751..4775)
                     /label=hm1100
                     /note="Cas9(BE4max) 1215aa btm ,25"
                     /note="color: ?; sequence: cccttctgcagttcgccggcagagg"
     primer_bind     complement(4947..4965)
                     /label=kn1063
                     /note="hCas9_4k_R"
                     /note="color: ?; sequence: attagcgtcggccaggatc"
     primer_bind     complement(5192..5211)
                     /label=Sy752
                     /note="color: ?; sequence: gtcacctcccagctgagaca"
     primer_bind     5211..5235
                     /label=al133
                     /note="nCas9-UGI-UGI,41"
                     /note="color: ?; sequence: 
                     agattatcaagaaaggcagcggcgggagcggcgggagcggg"
     CDS             5242..5490
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /note="color: #993366"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5521..5769
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /note="color: #993366"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     5770..5789
                     /label=Sy751
                     /note="color: ?; sequence: 
                     cagctgggaggtgactctggcggctcaaaaagaac"
     CDS             5812..5832
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     primer_bind     complement(5812..5828)
                     /label=hm1083
                     /note="NLS3'for T7SU,48"
                     /note="color: ?; sequence: 
                     gctcGAATTCGGATCCTCTAGTCAacccaccttcctcttcttcttggg"
     primer_bind     complement(5812..5828)
                     /label=al092
                     /note="Cas-UGI,35"
                     /note="color: ?; sequence: 
                     TTGGTCATggtacccaccttcctcttcttcttggg"
     primer_bind     complement(5812..5828)
                     /label=al004
                     /note="dCas9_XbaI,45"
                     /note="color: ?; sequence: 
                     tccttatccggagcctctagactacaccttcctcttcttcttggg"
     primer_bind     complement(5812..5828)
                     /label=hm498
                     /note="Cas9 NLS 3' btm w SphI,35"
                     /note="color: ?; sequence: 
                     ttaGCATGCtatcacaccttcctcttcttcttggg"
     primer_bind     complement(5812..5828)
                     /label=hm567
                     /note="Cas9 R691A 3' btm,45"
                     /note="color: ?; sequence: 
                     CGAATTCGGATCCTCTAGTTATCACACCTTCCTCTTCTTCTTGGG"
     primer_bind     complement(5812..5828)
                     /label=kn946
                     /note="Cas9-nosT_R"
                     /note="color: ?; sequence: 
                     tcgagctccctaactagtcaccttcctcttcttcttggg"
     primer_bind     complement(5813..5828)
                     /label=sy317
                     /note="Cas9 Rv mitchys vector"
                     /note="color: ?; sequence: caccttcctcttcttcttgg"
     primer_bind     complement(5813..5828)
                     /label=Sy389
                     /note="color: ?; sequence: ctacaccttcctcttcttcttgg"
     primer_bind     complement(5813..5828)
                     /label=Sy424
                     /note="GS linker SY20"
                     /note="color: ?; sequence: 
                     CCtCCGCCgaaattcaccttcctcttcttcttgg"
     primer_bind     complement(5813..5828)
                     /label=Sy421
                     /note="GS linker SY20"
                     /note="color: ?; sequence: 
                     cttctattgacttggcaccttcctcttcttcttgg"
     primer_bind     complement(5813..5828)
                     /label=Sy473
                     /note="color: ?; sequence: 
                     TAAACTTAAGCTTGCctacaccttcctcttcttcttgg"
     primer_bind     complement(5813..5828)
                     /label=kn1476
                     /note="Xho-Xba-Apa-NLS_R"
                     /note="color: ?; sequence: 
                     GACATAACTAATTACATGACTCGAGtctagaaccgggcccCACCTTCCTCTTCTTCTTG
                     G"
     primer_bind     complement(5814..5828)
                     /label=hm661
                     /note="Cas9-NLS_3' btm for GA w SUMOstar,44"
                     /note="color: ?; sequence: 
                     AAGGACGCGGTACCGCGGCCGCtcacaccttcctcttcttcttg"
     primer_bind     complement(5830..5849)
                     /label=al127
                     /note="color: ?; sequence: gtgatgatgaccggttagac"
     CDS             5841..5858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     5870..5888
                     /label=sy204
                     /note="vector Fw"
                     /note="color: ?; sequence: 
                     AACTGGGGCACAGCTAGgTTAAACCGCTGATCAGCCTCGACT"
     primer_bind     5876..5895
                     /label=sy313
                     /note="SY20 dual promoterp BGH polyA"
                     /note="color: ?; sequence: ATCAGCCTCGACTGTGCCTT"
     primer_bind     5876..5895
                     /label=Sy483
                     /note="color: ?; sequence: 
                     aCTCGAGAACCCAGCATCAGCCTCGACTGTGCCTT"
     primer_bind     5876..5895
                     /label=Sy478
                     /note="color: ?; sequence: 
                     tgataagagctccTCATCAGCCTCGACTGTGCCTT"
     primer_bind     complement(5881..5898)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /note="color: ?; sequence: TAGAAGGCACAGTCGAGG"
     polyA_signal    5887..6111
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     5887..5902
                     /label=al135
                     /note="nCas9,36"
                     /note="color: ?; sequence: 
                     GTCtgataagcggccgccccctgtgccttctagttg"
     primer_bind     complement(6075..6094)
                     /label=Sy769
                     /note="color: ?; sequence: TCCCCAGCATGCCTGCTATT"
     primer_bind     complement(6092..6111)
                     /label=al134
                     /note="nCas9/nCas9-UGI-UGI,50"
                     /note="color: ?; sequence: 
                     aatgtcaaCGCGTgggcccactagtgAATTccatagagcccaccgcatcc"
     primer_bind     complement(6100..6120)
                     /label=sy231
                     /note="SY4 ª@gX|]p BamHI Rv2"
                     /note="color: ?; sequence: catggatccCTCAGAAGCCATAGAGCCCA"
     primer_bind     complement(6100..6120)
                     /label=sy229
                     /note="SY4 ª@gX|]p EcoRI"
                     /note="color: ?; sequence: ctcgaattcCTCAGAAGCCATAGAGCCCA"
     primer_bind     complement(6100..6119)
                     /label=sy312
                     /note="SY20 dual promoterp BGH polyA OL292"
                     /note="color: ?; sequence: 
                     gcgactctaAccggtCTCAGAAGCCATAGAGCCCA"
     primer_bind     complement(6100..6119)
                     /label=sy239
                     /note="SY5 short"
                     /note="color: ?; sequence: 
                     TCCTGGCCTTTTGCTGCTCAGAAGCCATAGAGCCCA"
     primer_bind     complement(6103..6123)
                     /label=sy233
                     /note="1929 EGFP Fw with BamHI"
                     /note="color: ?; sequence: catggatccCCGCCTCAGAAGCCATAGAGC"
     primer_bind     complement(6103..6119)
                     /label=Sy785
                     /note="color: ?; sequence: 
                     CGAGCTCAAGCTTAAGTCTCAGAAGCCATAGAGC"
     primer_bind     complement(6107..6130)
                     /label=sy235
                     /note="SY15-3 RFP Rv BamHI"
                     /note="color: ?; sequence: 
                     catggatccGTTCTTTCCGCCTCAGAAGCCATA"
     primer_bind     complement(6108..6130)
                     /label=sy242
                     /note="SY6"
                     /note="color: ?; sequence: 
                     CCTGGCCTTTTGCTGTTCTTTCCGCCTCAGAAGCCAT"
     primer_bind     complement(6112..6136)
                     /label=hm381
                     /note="bGH3' for GA,40"
                     /note="color: ?; sequence: 
                     tccacagaattaattCAGCTGGTTCTTTCCGCCTCAGAAG"
     primer_bind     6112..6133
                     /label=al192
                     /note="""sy42, 26 without-iRFP"",50"
                     /note="color: ?; sequence: 
                     CCAAACTCATCAATGTATCTTAGCggcccttctgaggcggaaagaaccag"
     primer_bind     complement(6116..6140)
                     /label=hm411
                     /note="bGH pA 3' for hsvTK GA btm,25"
                     /note="color: ?; sequence: GCCCCAGCTGGTTCTTTCCGCCTCA"
     primer_bind     6165..6187
                     /label=kn381
                     /note="LacZA1_R"
                     /note="color: ?; sequence: 
                     ACGACGGCCAGTGAGTTCGAGCTCGGAACCTCGCGAATACATCTGGATATCGGATCACG
                     GGCCCGTCGACTACAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATA"
     primer_bind     complement(6166..6187)
                     /label=kn413
                     /note="Left-LacZ_F"
                     /note="color: ?; sequence: 
                     ATACCACTGGCGGTGATACGTATGCATATGACCATGATTACGCCAAGC"
     primer_bind     6166..6185
                     /label=kn683
                     /note="NotBspEBcl-T3_R"
                     /note="color: ?; sequence: 
                     cgtccggattgcggccgcttggcgtaatcatggtca"
     primer_bind     complement(6172..6187)
                     /label=kn372
                     /note="cro-SD-LacZ_F"
                     /note="color: ?; sequence: 
                     GTAATAGAGGAGGTTAGTATGACCATGATTACG"
     primer_bind     complement(6181..6202)
                     /label=kn584
                     /note="M13R-Long"
                     /note="color: ?; sequence: cacacaggaaacagctatgacc"
     primer_bind     complement(6182..6199)
                     /label=kn98
                     /note="Pex3intR_M13R"
                     /note="color: ?; sequence: 
                     ggtaatgtcatgttacctattgcacacttactgtattaaagattacgctaCAGGAAACA
                     GCTATGAC"
     primer_bind     complement(6182..6199)
                     /label=kn99
                     /note="Pex19intR_M13R"
                     /note="color: ?; sequence: 
                     ccacttttttttttcattttttaaagtgttatacttagttatgctctaggataatgaac
                     taCAGGAAACAGCTATGAC"
     primer_bind     complement(6182..6198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(6182..6198)
                     /label=hm708
                     /note="M13 rev,17"
                     /note="color: ?; sequence: caggaaacagctatgac"
     primer_bind     complement(6182..6198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
                     /note="color: ?; sequence: CAGGAAACAGCTATGAC"
     primer_bind     complement(6195..6217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
                     /note="color: ?; sequence: AGCGGATAACAATTTCACACAGG"
     primer_bind     6204..6220
                     /label=hm070
                     /note="hm014 target 44-2,44"
                     /note="color: ?; sequence: 
                     CGAGGTAGTAGGTTGTAACTAGAGGGGAATTGTTATCCGCTCAC"
     protein_bind    6206..6222
                     /label=lac operator
                     /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)."
                     /note="color: #31849b"
     promoter        complement(6230..6260)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 6230 .. 6236 / #ffffff / -10
                        2: 6237 .. 6254 / #ffffff
                        3: 6255 .. 6260 / #ffffff / -35"
     primer_bind     6234..6248
                     /label=kn2488
                     /note="tracr-in-UV5_R"
                     /note="color: ?; sequence: 
                     AACTTGCTATGCTGTTTTGAATGGTTccAACAAGATCATTATACGAGCCGGAAGC"
     protein_bind    6275..6296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     primer_bind     6285..6305
                     /label=kn1218
                     /note="ade1-HpaI-pUC19"
                     /note="color: ?; sequence: 
                     GTGGCCAGAGGTAAAGTTAGAGACATATATGAGGTAGACGCTGGTACGTTAACTCACAT
                     TAATTGCGTTG"
     primer_bind     6297..6313
                     /label=kn2239
                     /note="LacI_OL_R"
                     /note="color: ?; sequence: 
                     gagataatttatcaccgcAGATGGTTATCGattgcgttgcgctcact"
     primer_bind     complement(6306..6322)
                     /label=kn2353
                     /note="LacI_end_F"
                     /note="color: ?; sequence: aaagcgggcagtgagcg"
     primer_bind     complement(6309..6328)
                     /label=Sy825
                     /note="color: ?; sequence: gactggaaagcgggcagtga"
     primer_bind     6336..6358
                     /label=hm036
                     /note="pEX-A2J1 BsaI F,41"
                     /note="color: ?; sequence: 
                     tgaTCTAGAgagaccgggCTGTCGTGCCAGCTGCATTAATG"
     primer_bind     complement(6377..6403)
                     /label=hm354
                     /note="sy5 Nco-frag1 to remove SapI btm,35"
                     /note="color: ?; sequence: 
                     GCGGActcGAGCGCCCAATACGCAAACcgcctctc"
     primer_bind     6405..6423
                     /label=hm355
                     /note="sy5 Nco-frag2 to remove SapI top,29"
                     /note="color: ?; sequence: GGCGCTCgagTCCGCTTCCTCGCTCACTG"
     primer_bind     6411..6430
                     /label=kn707
                     /note="ColE-Bam-Spe-Sac1_R"
                     /note="color: ?; sequence: 
                     ATTTAAAAGCAGCGGGAGCTCagactagtcggatcctcgctcactgactcgct"
     primer_bind     complement(6413..6430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: ?; sequence: AGCGAGTCAGTGAGCGAG"
     primer_bind     6450..6467
                     /label=kn1622
                     /note="pKNT19-ClaSpeNgo_F"
                     /note="color: ?; sequence: 
                     cTGCATCGATGAAGATTCTTGCTCactagtgcCGGCGAGCGGTATCAGCT"
     primer_bind     6486..6505
                     /label=hm734
                     /note="ori down stream,20"
                     /note="color: ?; sequence: CGGTTATCCACAGAATCAGG"
     primer_bind     complement(6512..6530)
                     /label=hm626
                     /note="CMV up top w dw bGHpA (hm463),39"
                     /note="color: ?; sequence: 
                     atcgttccactgagcgtcagtcacatgttctttcctgcg"
     primer_bind     6520..6538
                     /label=hm408
                     /note="upstream p15A ori in p909 top for ga,39"
                     /note="color: ?; sequence: 
                     GTTGTAAAACGACGGCCAGTAGAACATGTGAGCAAAAGG"
     primer_bind     6521..6538
                     /label=hm412
                     /note="Amp vec for hsvTK series GA btm,36"
                     /note="color: ?; sequence: 
                     GAAAGAACCAGCTGGGGCGAACATGTGAGCAAAAGG"
     primer_bind     6525..6543
                     /label=hm546
                     /note="Amp-ori casette for ga w T7-MS2 btm,40"
                     /note="color: ?; sequence: 
                     CTGGGCTGCTCGCCACAGGTTATGTGAGCAAAAGGCCAGC"
     primer_bind     6530..6548
                     /label=kn1269
                     /note="pTDH3-Nhe-p426_R"
                     /note="color: ?; sequence: 
                     GGCAGTATTGATAATGAattcgctagcaaaaggccagcaaaag"
     primer_bind     6535..6554
                     /label=hm593
                     /note="ori for HA-R,40"
                     /note="color: ?; sequence: 
                     ggggttcctggctctgctctAAGGCCAGCAAAAGGCCAGG"
     primer_bind     6535..6554
                     /label=hm620
                     /note="aavs1 HA I-SceI 1,58"
                     /note="color: ?; sequence: 
                     ggggttcctggctctgctctTAGGGATAACAGGGTAATAAGGCCAGCAAAAGGCCAGG"
     primer_bind     6537..6563
                     /label=Sy681
                     /note="Co[XHEK2 1"
                     /note="color: ?; sequence: 
                     GAACACAAAGCATAGACTGCGGGCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6539..6563
                     /label=Sy826
                     /note="color: ?; sequence: 
                     gcccgctttccagtccagcaaaaggccaggaaccgtaaa"
     primer_bind     6539..6563
                     /label=Sy700
                     /note="color: ?; sequence: 
                     CCTACAGTCTATTCTCTTTGCTCCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6539..6563
                     /label=Sy698
                     /note="color: ?; sequence: 
                     CCCGCAGTCTATGCTTTGTGTTCCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6539..6563
                     /label=Sy552
                     /note="color: ?; sequence: 
                     ACATTGATTATTGACCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6540..6563
                     /label=Sy729
                     /note="color: ?; sequence: 
                     CCGCAAATGCTTCTAGGCGGACTCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6540..6563
                     /label=sy238
                     /note="SY5 short"
                     /note="color: ?; sequence: CAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6540..6563
                     /label=Sy679
                     /note="Co[XsgRNA2"
                     /note="color: ?; sequence: 
                     GAGCAAAGAGAATAGACTGTAGGCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6540..6563
                     /label=Sy592
                     /note="color: ?; sequence: 
                     GACCCCCTCCACCCCGCCTCCGGCAGCAAAAGGCCAGGAACCGTAAA"
     primer_bind     6546..6568
                     /label=kn420
                     /note="Gal1R-pBR322_R"
                     /note="color: ?; sequence: 
                     GTATATGGATATGTATATGGTGGTAATGCCATGTAAGGCCAGGAACCGTAAAAAGGC"
     primer_bind     6560..6578
                     /label=kn2479
                     /note="ori-pacI_R"
                     /note="color: ?; sequence: 
                     ctgagtattcccacagttaattaaaaaggccgcgttgctg"
     primer_bind     6563..6581
                     /label=hm386
                     /note="sv40pa ori top for ga,36"
                     /note="color: ?; sequence: 
                     CGTCGACCTCTAGCTAGAAAGGCCGCGTTGCTGGCg"
     primer_bind     6580..6599
                     /label=kn2403
                     /note="SNR-Spe-Ori_R"
                     /note="color: ?; sequence: 
                     caaagactagtggcgcgccgtttttccataggctccgc"
     primer_bind     complement(6581..6600)
                     /label=kn746
                     /note="P15AendR"
                     /note="color: ?; sequence: GGCGGAGCCTATGGAAAAAC"
     primer_bind     6581..6599
                     /label=kn1590
                     /note="Km_gRNAvector_2"
                     /note="color: ?; sequence: 
                     aggccggccatagggataacagggtaatgttaaccggtttttccataggctccgc"
     rep_origin      complement(6584..7172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     6584..6606
                     /label=hm596
                     /note="ori 3' btm,23"
                     /note="color: ?; sequence: tttccataggctccgcccccctg"
     primer_bind     complement(6625..6643)
                     /label=kn709
                     /note="ColE1"
                     /note="color: ?; sequence: cacctctgacttgagcgtc"
     primer_bind     complement(6664..6683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: ?; sequence: GGGAAACGCCTGGTATCTTT"
     primer_bind     6862..6887
                     /label=sy208
                     /note="Amp_ori_Rv"
                     /note="color: ?; sequence: ACCGCTGCGCCTTATCCGGTAACTAT"
     primer_bind     7007..7045
                     /label=hm732
                     /note="ColE1 ori for C>T mut btm,39"
                     /note="color: ?; sequence: 
                     CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGC"
     primer_bind     complement(7007..7024)
                     /label=hm733
                     /note="ColE1 ori for C>T mut top,18"
                     /note="color: ?; sequence: CTTCTAGTGTAGCCGTAG"
     primer_bind     7097..7116
                     /label=kn2444
                     /note="ori_F"
                     /note="color: ?; sequence: caaacaaaccaccgctggta"
     primer_bind     complement(7151..7172)
                     /label=kn2443
                     /note="ori_R"
                     /note="color: ?; sequence: ttgagatcctttttttctgcgc"
     primer_bind     complement(7170..7193)
                     /label=kn875
                     /note="ChimerSpeNotColE_R"
                     /note="color: ?; sequence: 
                     GGTCATTAAAACtagtagcgccggcgagaaaagatcaaaggatcttcttg"
     primer_bind     7200..7221
                     /label=sy109
                     /note="1935#2 for IRESmEGFP Fw,59"
                     /note="color: ?; sequence: TCTGACGCTCAGTGGAACGAAA"
     primer_bind     7219..7241
                     /label=kn2547
                     /note="BsaX-DR_F"
                     /note="color: ?; sequence: 
                     gCcggagnacgcgtgcGTTTAAGAGCTATGCTGTTTTGAATGGTCCCAAAACTCACGTT
                     AAGGGATTTTG"
     primer_bind     7227..7255
                     /label=kn879
                     /note="ampT-age1-p15A_R"
                     /note="color: ?; sequence: 
                     CATCTTCCAGGAAATCTCcgttaagggataccggtcatgagattatc"
     primer_bind     complement(7227..7248)
                     /label=hm788
                     /note="p15A ori up,44"
                     /note="color: ?; sequence: 
                     TGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACG"
     primer_bind     7235..7261
                     /label=kn2520
                     /note="Amp-Cla_R"
                     /note="color: ?; sequence: 
                     cgcaacgcaatCgattttggtcatgagattatcaaaaag"
     primer_bind     7237..7262
                     /label=kn874
                     /note="AmpAgeXbaChimr_F"
                     /note="color: ?; sequence: 
                     CCtctagactaccggttttggtcatgagattatcaaaaagg"
     primer_bind     7241..7265
                     /label=kn976
                     /note="amp-age_R"
                     /note="color: ?; sequence: 
                     catgatcactagatcctaggactctagattaccggtcatgagattatcaaaaaggatc"
     primer_bind     7244..7261
                     /label=hm752
                     /note="Amp-ori for ga with 1105 fw,41"
                     /note="color: ?; sequence: 
                     TATGGCGTGCTGCTAGCcgatggcatgagattatcaaaaag"
     CDS             complement(7343..8203)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 7343 .. 8134 / #ccffcc
                        2: 8135 .. 8203 / #ccffcc / signal sequence
                     Cleavage site after base 8134"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7346..7365
                     /label=kn189
                     /note="BLF2_SNP_R"
                     /note="color: ?; sequence: 
                     ctgcagcggccgctactagtCCAATGCTTAATCAGTGAGG"
     primer_bind     complement(7392..7411)
                     /label=hm516
                     /note="AmpR 265th aa fw for seq,20"
                     /note="color: ?; sequence: caggcaactatggatgaacg"
     primer_bind     complement(7426..7447)
                     /label=sy207
                     /note="Amp_ori_Fw"
                     /note="color: ?; sequence: CCCTCCCGTATCGTAGTTATCT"
     primer_bind     complement(7468..7497)
                     /label=kn705
                     /note="ampR-Bsa1KO_F"
                     /note="color: ?; sequence: gtgagcgtggttctcgcggtatcattgcag"
     primer_bind     7471..7502
                     /label=kn704
                     /note="ampR-Bsa1KO_R"
                     /note="color: ?; sequence: 
                     caatgataccgcgagaaccacgctcaccggct"
     primer_bind     complement(7545..7568)
                     /label=hm519
                     /note="AmpR 212nd aa fw for seq,24"
                     /note="color: ?; sequence: taaagttgcaggaccacttctgcg"
     primer_bind     complement(7599..7619)
                     /label=kn188
                     /note="BLF2_ENX_F"
                     /note="color: ?; sequence: 
                     tgaattcgcggccgcttctagaCTACTTACTCTAGCTTCCCG"
     primer_bind     7622..7643
                     /label=kn185
                     /note="BLF1_SNP_R"
                     /note="color: ?; sequence: 
                     ctgcagcggccgctactagtGCCAGTTAATAGTTTGCGCaac"
     primer_bind     7763..7782
                     /label=hm656
                     /note="AmpR mdl btm,20"
                     /note="color: ?; sequence: ggttagctccttcggtcctc"
     primer_bind     7966..7985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: ?; sequence: ATAATACCGCGCCACATAGC"
     primer_bind     complement(8107..8129)
                     /label=kn184
                     /note="BLF1_ENX_F"
                     /note="color: ?; sequence: 
                     tgaattcgcggccgcttctagaGAAACGCTGGTGAAAGTAAAAG"
     primer_bind     8115..8136
                     /label=hm518
                     /note="AmpR 30th aa rv for seq,22"
                     /note="color: ?; sequence: ttcaccagcgtttctgggtgag"
     primer_bind     8142..8161
                     /label=kn398
                     /note="B-Lactamase_50_R"
                     /note="color: ?; sequence: ACAGGAAGGCAAAATGCCGC"
     primer_bind     complement(8184..8203)
                     /label=kn397
                     /note="B-lactamase_F"
                     /note="color: ?; sequence: ATGAGTATTCAACATTTCCG"
     promoter        complement(8204..8308)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     8241..8260
                     /label=kn708
                     /note="AmpP_R"
                     /note="color: ?; sequence: ggttattgtctcatgagcgg"
     primer_bind     complement(8271..8291)
                     /label=hm723
                     /note="KmR dw btm,36"
                     /note="color: ?; sequence: 
                     ctaaaagccagataaTTTATTTTTCTAAATACATTC"
     primer_bind     complement(8282..8308)
                     /label=al106
                     /note="gRNA vector,27"
                     /note="color: ?; sequence: cgcggaacccctatttgtttatttttc"
     primer_bind     complement(8292..8309)
                     /label=hm665
                     /note="AmpR pro upstream top,38"
                     /note="color: ?; sequence: 
                     ctgtcgcacaaccatgGCTAGcgcggaacccctatttg"
     primer_bind     complement(8292..8309)
                     /label=kn2434
                     /note="Leu-IsceAvr-Amp_F"
                     /note="color: ?; sequence: 
                     cacattaccctgttatccctaggcgcggaacccctatttg"
     primer_bind     complement(8292..8308)
                     /label=al100
                     /note="SaCas9 vector,45"
                     /note="color: ?; sequence: 
                     cttatataagtacgctagcgacgtcgaccgcggaacccctatttg"
     primer_bind     complement(8292..8308)
                     /label=hm517
                     /note="AmpR pro 5' fw for seq,17"
                     /note="color: ?; sequence: cgcggaacccctatttg"
     primer_bind     8302..8327
                     /label=hm780
                     /note="gRNA cassette btm for GA w/ pET_Ago+MF,39"
                     /note="color: ?; sequence: 
                     gcatcaggcgctcttccgctCACATTTCCCCGAAAAGTG"
     primer_bind     8305..8329
                     /label=kn1154
                     /note="pKNT19_aat2_F"
                     /note="color: ?; sequence: 
                     cctGTGTATAACTCACTACTTTAGTCAGTTCCgacgtgcACATTTCCCCGAAAAGTGCC
                     "
     primer_bind     8309..8334
                     /label=hm910
                     /note="T7 tt for GA w/ bom,44"
                     /note="color: ?; sequence: 
                     gaaaataccgcatcaggcCACATTTCCCCGAAAAGTGCCACCTG"
     primer_bind     complement(8314..8332)
                     /label=kn2519
                     /note="Amp-XmaI_F"
                     /note="color: ?; sequence: 
                     gaagtcagccccgggtggcacttttcggggaa"
     primer_bind     complement(8315..8332)
                     /label=kn2073
                     /note="AgeI-H1p_R"
                     /note="color: ?; sequence: 
                     GACGTCAGCGTTCGAATTggtacaccggtggcacttttcgggGA"
     primer_bind     complement(8316..8334)
                     /label=hm387
                     /note="amp pro for ga btm,36"
                     /note="color: ?; sequence: 
                     ATCTGGCCCGTACATCGCAGGTGGCACTTTTCGGGG"
     primer_bind     complement(8316..8334)
                     /label=hm029
                     /note="rrn-downKm ga FOR,38"
                     /note="color: ?; sequence: 
                     GACGGATGGCCTTTTTGCGcaggtggcacttttcgggg"
     primer_bind     complement(8316..8334)
                     /label=hm545
                     /note="Amp-ori casette for ga w T7-MS2 top,39"
                     /note="color: ?; sequence: 
                     gggtcttgaggggttttttgCAGGTGGCACTTTTCGGGG"
     primer_bind     complement(8317..8334)
                     /label=hm020
                     /note="pET24 downKm ga FOR,42"
                     /note="color: ?; sequence: CCAGAGCTGCcaggtggcacttttcggg"
     primer_bind     complement(8318..8334)
                     /label=kn909
                     /note="aatL2-amp_F"
                     /note="color: ?; sequence: 
                     ttcttgtacaaagttgggcggccgcggatcccaggtggcacttttcgg"
     primer_bind     complement(8320..8339)
                     /label=kn853
                     /note="rrnBt-Nsi-Aat2_R"
                     /note="color: ?; sequence: 
                     GGCCTTTCGTTTTATctgttgtttgCGGCCGatcatgcatgagacgtcaggtggcactt
                     ttc"
     primer_bind     8330..8347
                     /label=kn1311
                     /note="pcDNA01_F"
                     /note="color: ?; sequence: ACCTGACGTCGACGGATC"
     primer_bind     complement(8387..8406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="color: ?; sequence: CGGCATCAGAGCAGATTGTA"
     primer_bind     complement(8388..8407)
                     /label=kn1198
                     /note="Ura3pro_F"
                     /note="color: ?; sequence: gcggcatcagagcagattgt"
     primer_bind     complement(8393..8412)
                     /label=kn374
                     /note="tonBt-LacZA_R"
                     /note="color: ?; sequence: 
                     CAATTGGTTCAGACGCTACTTTTTTACTATGCGGCATCAGAGCAG"
     primer_bind     complement(8419..8440)
                     /label=sy101
                     /note="SY1-3 Rv EF1at¯O¦,58"
                     /note="color: ?; sequence: ACACAAGCAGGGAGCAGATACT"
     primer_bind     complement(8486..8507)
                     /label=sy183
                     /note="at004"
                     /note="color: ?; sequence: ATTGTCGGTCAAGCCTTGCCTT"
     primer_bind     8487..8508
                     /label=sy182
                     /note="at003"
                     /note="color: ?; sequence: AGGCAAGGCTTGACCGACAATT"
     primer_bind     8525..8545
                     /label=sy35
                     /note="EF1a promoter Fw +/-eEoverlap^X,59"
                     /note="color: ?; sequence: 
                     ATATCTGGCCCGTACATCGtagggttaggcgttttgcgct"
     primer_bind     join(8569..8577,1..16)
                     /label=Sy332
                     /note="SY4 gRNA²«"
                     /note="color: ?; sequence: 
                     GGCTTGACCGACAATACGCGTTGACATTGATTATTGAC"
     primer_bind     complement(join(8570..8577,1..34))
                     /label=hm373
                     /note="MS2 CMVpro btm long,42"
                     /note="color: ?; sequence: 
                     GATTACTATTAATAACTAGTCAATAATCAATGTCAACGCGTA"
     primer_bind     join(8570..8577,1..16)
                     /label=Sy395
                     /note="color: ?; sequence: 
                     gggtaacgccagggtACGCGTTGACATTGATTATTGAC"
     primer_bind     complement(join(8570..8577,1..16))
                     /label=hm372
                     /note="MS2 CMVpro btm short,24"
                     /note="color: ?; sequence: GTCAATAATCAATGTCAACGCGTA"
     primer_bind     join(8570..8577,1..15)
                     /label=kn2215
                     /note="CjSg_F"
                     /note="color: ?; sequence: 
                     TACAATCCCCTAAAACCGCTTTTTTACGCGTGACATTGATTATTGA"
     primer_bind     join(8570..8577,1..15)
                     /label=kn2208
                     /note="sgRNA_F2"
                     /note="color: ?; sequence: 
                     TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTACGCGTTGACATTGATTATTGA"
     primer_bind     join(8570..8577,1..7)
                     /label=Sy564
                     /note="color: ?; sequence: 
                     aacttgttggcgagaTTTTTTTACGCGTTGACATTG"
     primer_bind     join(8571..8577,1..16)
                     /label=sy301
                     /note="CMV promoter@without gRNA"
                     /note="color: ?; sequence: 
                     ttgacggggaaagccACGCGTTGACATTGATTATTGAC"
     primer_bind     join(8571..8577,1..16)
                     /label=hm1070
                     /note="up ori,44"
                     /note="color: ?; sequence: 
                     tgtgAATTcactagtgggcccACGCGTTGACATTGATTATTGAC"
     primer_bind     join(8571..8577,1..13)
                     /label=hm413
                     /note="CMV pro 5' for pCMV-hsvTK GA top,38"
                     /note="color: ?; sequence: 
                     GCCACCTGgcagctctggACGCGTTGACATTGATTATT"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcgacaag
      721 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac
      781 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
      841 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
      901 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
      961 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     1021 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     1081 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     1141 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     1201 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     1261 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     1321 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     1381 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
     1441 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     1501 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     1561 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     1621 gacatcctga gagtgaacac cgaaatcacc aaggcccccc tgagcgcctc tatgatcaag
     1681 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     1741 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     1801 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     1861 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     1921 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     1981 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     2041 ctgaccttcc gcattcccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     2101 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     2161 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     2221 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     2281 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     2341 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     2401 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     2461 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     2521 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     2581 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     2641 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     2701 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     2761 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     2821 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     2881 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     2941 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     3001 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     3061 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     3121 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     3181 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     3241 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     3301 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     3361 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     3421 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     3481 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     3541 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     3601 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     3661 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     3721 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     3781 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     3841 aactttttca agaccgagat taccctggcc aactcacacc gctgctacgt gctgtttgag
     3901 ttgaagcgaa ggggcgaaag aagggcttgc ttttggggct atgccgtcaa caagccccaa
     3961 agtggcaccg agagaggaat acacgctgag atattcagta tccgaaaggt ggaagagtat
     4021 cttcgggata atcctgggca gtttacgatc aactggtatt ccagctggag tccttgcgct
     4081 gattgtgccg agaaaattct ggaatggtat aatcaggaac ttcggggaaa cgggcacaca
     4141 ttgaaaatcg aagcctgcaa gctgtactac gagaagaatg cccggaacca gataggactc
     4201 cagaatctga gggacaatgg tgtaggcctg aacgtgggtg gaggtggcag tgatgtcggc
     4261 ggaggctccg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     4321 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     4381 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     4441 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     4501 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     4561 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     4621 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     4681 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     4741 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     4801 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     4861 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     4921 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     4981 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     5041 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     5101 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     5161 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggtga cagcggcggg
     5221 agcggcggga gcggggggag cactaatctg agcgacatca ttgagaagga gactgggaaa
     5281 cagctggtca ttcaggagtc catcctgatg ctgcctgagg aggtggagga agtgatcggc
     5341 aacaagccag agtctgacat cctggtgcac accgcctacg acgagtccac agatgagaat
     5401 gtgatgctgc tgacctctga cgcccccgag tataagcctt gggccctggt catccaggat
     5461 tctaacggcg agaataagat caagatgctg agcggaggat ccggaggatc tggaggcagc
     5521 accaacctgt ctgacatcat cgagaaggag acaggcaagc agctggtcat ccaggagagc
     5581 atcctgatgc tgcccgaaga agtcgaagaa gtgatcggaa acaagcctga gagcgatatc
     5641 ctggtccata ccgcctacga cgagagtacc gacgaaaatg tgatgctgct gacatccgac
     5701 gccccagagt ataagccctg ggctctggtc atccaggatt ccaacggaga gaacaaaatc
     5761 aaaatgctgt ctggcggctc aaaaagaacc gccgacggca gcgaattcga gcccaagaag
     5821 aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
     5881 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5941 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     6001 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     6061 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     6121 cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat
     6181 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6241 ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg
     6301 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6361 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6421 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6481 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6541 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6601 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6661 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6721 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6781 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6841 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6901 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6961 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7021 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7081 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7141 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7201 ctgacactca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7261 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7321 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7381 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7441 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7501 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7561 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7621 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7681 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7741 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7801 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7861 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7921 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7981 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8041 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8101 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8161 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8221 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8281 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     8341 acggatcggg agatcgattt cccgatcccc tagggtcgac tctcagtaca atctgctctg
     8401 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     8461 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     8521 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
