LOCUS       Exported                9131 bp ds-DNA     circular SYN 25-JUN-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 9131)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Mar 18, 2019 from SnapGene 4.3.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9131
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     1..44
                     /label=F236
     misc_feature    1..19
                     /label=Promoter PpflB
     primer_bind     20..70
                     /label=LC1154
                     /note="pLC157_BsaI"
     primer_bind     20..70
                     /label=LC295
                     /note="sgRNAlib"
     primer_bind     20..43
                     /label=ES99
                     /note="ctrole-F"
     primer_bind     24..77
                     /label=V166
                     /note="R43-F"
     primer_bind     24..70
                     /label=LC576_pLtetBsaIsg
                     /note="long oligo for pLtetO-1 BsaI sgRNA construct 
                     (pLC63)"
     primer_bind     24..70
                     /label=LC384
                     /note="LC7_oligo"
     primer_bind     24..49
                     /label=LC860
                     /note="sgRNA_R2"
     primer_bind     complement(24..47)
                     /label=ES98
                     /note="ctrole-R"
     primer_bind     24..44
                     /label=F145
     misc_feature    24..43
                     /label=Control spacer with 2 BsaI
     misc_feature    24..34
                     /label=Control spacer with 2 BsaI
     primer_bind     42..66
                     /label=V92
                     /note="pAV4topAV6_F"
     primer_bind     43..70
                     /label=LC462
                     /note="depG_oligo"
     primer_bind     43..70
                     /label=LC312
                     /note="eamBsg"
     primer_bind     43..70
                     /label=LC311
                     /note="garDsg"
     misc_RNA        44..126
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the CRISPR/Cas9 system"
     misc_RNA        44..119
                     /label=gRNA scaffold (shorter version)
                     /note="guide RNA scaffold for the CRISPR/Cas9 system"
     primer_bind     complement(44..103)
                     /label=LC297
                     /note="LC_array_R"
     primer_bind     complement(44..77)
                     /label=LC638
                     /note="DBlib_P1_R_8N"
     primer_bind     complement(44..76)
                     /label=LC867
                     /note="DB_P1R_N6"
     primer_bind     complement(44..76)
                     /label=LC637
                     /note="DBlib_P1_R_7N"
     primer_bind     complement(44..76)
                     /label=LC869
                     /note="DB_P1R_N8"
     primer_bind     complement(44..75)
                     /label=LC868
                     /note="DB_P1R_N7"
     primer_bind     complement(44..74)
                     /label=LC866
                     /note="DB_P1R_N5"
     primer_bind     complement(44..74)
                     /label=LC615
                     /note="DBlib_P1_R"
     primer_bind     complement(44..73)
                     /label=LC636
                     /note="DBlib_P1_R_5N"
     primer_bind     complement(44..72)
                     /label=LC864
                     /note="DB_P1R_N3"
     primer_bind     complement(44..72)
                     /label=LC635
                     /note="DBlib_P1_R_4N"
     primer_bind     complement(44..72)
                     /label=LC865
                     /note="DB_P1R_N4"
     primer_bind     complement(44..71)
                     /label=LC634
                     /note="DBlib_P1_R_3N"
     primer_bind     complement(44..71)
                     /label=LC716
                     /note="DB_seq_R1"
     misc_feature    44..70
                     /label=Downstream sgRNA lib
     primer_bind     44..70
                     /label=V65
                     /note="spacer-scaffold-F"
     primer_bind     44..70
                     /label=LC1168
                     /note="pLC157_ibaG"
     primer_bind     44..70
                     /label=LC1157
                     /note="pLC157_secA"
     primer_bind     44..70
                     /label=LC463
                     /note="pal_oligo"
     primer_bind     44..70
                     /label=LC293
                     /note="sgRNAbblib_F"
     primer_bind     44..70
                     /label=LC313
                     /note="mhpRsg"
     primer_bind     44..70
                     /label=LC298
                     /note="LacZ1sg"
     primer_bind     complement(44..68)
                     /label=LC412
                     /note="sgRNA_R1"
     primer_bind     44..66
                     /label=LC1029
                     /note="ACCCA_non1_5F"
     primer_bind     44..66
                     /label=LC1027
                     /note="ACCCA_non1_10F"
     primer_bind     44..66
                     /label=B569
                     /note="rep5m_sp-F"
     primer_bind     44..66
                     /label=B562
                     /note="sgRNA-kansp-F"
     primer_bind     complement(63..84)
                     /label=LC877
                     /note="tracrRNA"
     primer_bind     complement(76..95)
                     /label=BG72
                     /note="psgRNAcos_qPCR_R"
     primer_bind     complement(107..127)
                     /label=V167
                     /note="R43-R"
     primer_bind     complement(110..140)
                     /label=LC271
                     /note="Cas9sgE2_R"
     primer_bind     complement(110..128)
                     /label=LC344
                     /note="sgphiattB_R"
     primer_bind     complement(110..128)
                     /label=LC335
                     /note="sgPhiC31P3R"
     primer_bind     complement(110..128)
                     /label=LC381
                     /note="Tracr_F2"
     primer_bind     complement(110..127)
                     /label=V66
                     /note="prefix-scaffold-R"
     primer_bind     complement(111..128)
                     /label=LC699
                     /note="pLC86insert_R"
     primer_bind     complement(111..128)
                     /label=LC405
                     /note="sgRNA_RF"
     primer_bind     complement(111..128)
                     /label=LC279
                     /note="sgRNA_R"
     primer_bind     complement(144..184)
                     /label=F219
     primer_bind     complement(144..183)
                     /label=F146
     primer_bind     complement(144..169)
                     /label=F197
     primer_bind     complement(144..168)
                     /label=F191
     primer_bind     154..193
                     /label=F144
     primer_bind     155..193
                     /label=F217
     terminator      164..168
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     169..207
                     /label=F96
     primer_bind     complement(241..280)
                     /label=F232
     primer_bind     251..290
                     /label=F234
     primer_bind     266..290
                     /label=LC929
                     /note="pLC110_FF1"
     primer_bind     266..290
                     /label=LC600_pLC67RP4_F
     primer_bind     266..290
                     /label=B148
                     /note="PstI-SW-R"
     primer_bind     266..284
                     /label=B758
                     /note="Csn2-R"
     primer_bind     281..297
                     /label=V97
                     /note="pAV7_vector_R"
     misc_feature    complement(292..548)
                     /label=RP4 transfer origin
     primer_bind     293..315
                     /label=B380
                     /note="(pCas9)-oriT-R"
     oriT            294..403
                     /note="incP origin of transfer"
     primer_bind     294..308
                     /label=FR173
                     /note="Adds oriT"
     primer_bind     complement(313..331)
                     /label=LC1195
                     /note="pLC159_F2R"
     primer_bind     complement(355..373)
                     /label=SW_F
                     /note="from pSW23t_AttP"
     primer_bind     complement(382..405)
                     /label=FR174
                     /note="Adds oriT"
     primer_bind     complement(520..542)
                     /label=B381
                     /note="(pCas9)-oriT-F"
     primer_bind     complement(548..585)
                     /label=F235
     primer_bind     complement(551..575)
                     /label=LC599_pLC67RP4_R
     primer_bind     complement(551..569)
                     /label=LC930
                     /note="pLC110_FR1"
     primer_bind     556..596
                     /label=F233
     promoter        complement(557..569)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(592..615)
                     /label=AB269
                     /note="seq_pLZ_circularize_R"
     primer_bind     640..673
                     /label=ACV80
                     /note="NHEJpAB62_Fw"
     primer_bind     986..1008
                     /label=W326
                     /note="WJ"
     primer_bind     complement(1305..1326)
                     /label=W459
                     /note="WJ"
     primer_bind     1514..1535
                     /label=W460
                     /note="WJ"
     primer_bind     1762..1784
                     /label=W354
                     /note="WJ"
     CDS             1902..2597
                     /codon_start=1
                     /label=repA
                     /translation="MAIKNTKARNFGFLLYPDSIPNDWKEKLESLGVSMAVSPLHDMDE
                     KKDKDTWNSSDVIRNGKHYKKPHYHVIYIARNPVTIESVRNKIKRKLGNSSVAHVEILD
                     YIKGSYEYLTHESKDAIAKNKHIYDKKDILNINDFDIDRYITLDESQKRKLKNLLLDIV
                     DDYNLVNTKDLMAFIRLRGAEFGILNTNDVKDIVSTNSSAFRLWFEGNYQCGYRASYAK
                     VLDAETGEIK"
     primer_bind     complement(2858..2883)
                     /label=W355
                     /note="WJ"
     CDS             complement(2887..3558)
                     /codon_start=1
                     /label=spec
                     /translation="MFGSGVESGLKPNSDLDFLVVVSEPLTDQSKEILIQKIRPISKKI
                     GDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYGEWLQELYEQGYIPQKELNSDLTIMLY
                     QAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMDSSEELIDNYQDDETNSILTLCRMILT
                     MDTGKIIPKDIAGNAVAESSPLEHRERILLAVRSYLGENIEWTNENVNLTINYLNNRLK
                     KL"
     primer_bind     complement(2989..3004)
                     /label=oGR394
                     /note="PCR_spec_F9_F"
     primer_bind     complement(3013..3033)
                     /label=oGR479
                     /note="PCR pRG76 spc amyE B lacZ"
     primer_bind     3477..3496
                     /label=oGR393
                     /note="PCR_amyE_front_spec_R"
     primer_bind     complement(3642..3666)
                     /label=W341
                     /note="WJ"
     primer_bind     complement(3860..3903)
                     /label=F216
     primer_bind     complement(3860..3884)
                     /label=F143
     primer_bind     3869..3912
                     /label=F218
     primer_bind     3884..3912
                     /label=F190
     primer_bind     3884..3909
                     /label=F7
                     /note="(pC194)-tetR-F"
     primer_bind     3884..3909
                     /label=F100
                     /note="pAS10 tetR-pLZ12"
     primer_bind     3884..3909
                     /label=B534 - (pE194)tetR-F
                     /note="from pKL55_iTet_RC12"
     primer_bind     3885..3904
                     /label=AB198
                     /note="pDB271_(pLZ)_F"
     primer_bind     3885..3902
                     /label=D014
                     /note="tet F"
     primer_bind     3885..3897
                     /label=ACV67
                     /note="pAB62_tetRfw"
     primer_bind     complement(3887..3911)
                     /label=ACV65
                     /note="pAB62_BB_rv"
     primer_bind     3887..3909
                     /label=VM9
                     /note="pDB71-backbone-Fwd"
     primer_bind     complement(3889..3913)
                     /label=ACV43
                     /note="pAB41_remove_tetR_Rv1"
     primer_bind     complement(3893..3913)
                     /label=AB213
                     /note="pAB41_remove_tetR_R"
     primer_bind     3915..3936
                     /label=ES76
                     /note="ptet_pACV02_primer5"
     CDS             complement(3916..4542)
                     /codon_start=1
                     /label=tetR
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3916..3953)
                     /label=ACV93
                     /note="pAB78_bb"
     primer_bind     3916..3944
                     /label=ACV5
                     /note="itet_pZS25_pTet_RecET_Fw"
     primer_bind     3916..3939
                     /label=LC100
                     /note="pITCas9_F"
     primer_bind     3916..3935
                     /label=LC653
                     /note="Tet_F1"
     primer_bind     3918..3942
                     /label=V64
                     /note="tracr-dCas9-F"
     primer_bind     4018..4043
                     /label=ACV44
                     /note="pAB41_remove_tetR_Fw2"
     primer_bind     complement(4414..4440)
                     /label=ACV45
                     /note="pAB41_remove_tetR_Rv2"
     primer_bind     4526..4559
                     /label=ES60
                     /note="dCas9_primer4"
     primer_bind     complement(4526..4559)
                     /label=ES57
                     /note="pACV5_primer1"
     primer_bind     4548..4576
                     /label=ACV42
                     /note="pAB41_remove_tetR_Fw1"
     primer_bind     4548..4576
                     /label=AB212
                     /note="pAB41_remove_tetR_F"
     primer_bind     4571..4608
                     /label=B697
                     /note="(pAZ1-bdk)pAS10-F"
     primer_bind     4576..4609
                     /label=AB18 (2)
                     /note="(pDB114)NHEJ ptet-F"
     primer_bind     4576..4609
                     /label=AB18
                     /note="(pDB114)NHEJ ptet-F"
     primer_bind     4604..4626
                     /label=B693
                     /note="GG71"
     primer_bind     4604..4626
                     /label=F98
                     /note="pAS10 ptet-pLZ12"
     primer_bind     complement(4616..4665)
                     /label=F102
                     /note="Ptet NNN (pAS10)"
     primer_bind     4626..4675
                     /label=F101
                     /note="NNN Ptet (pAS10)"
     misc_feature    4644..4648
                     /label=-35
     misc_feature    4650..4657
                     /label=rep
     primer_bind     complement(4655..4680)
                     /label=F8
                     /note="(bdk)-ptet-R"
     primer_bind     complement(4656..4678)
                     /label=B669
                     /note="protAsp-R"
     primer_bind     complement(4658..4681)
                     /label=B557
                     /note="sgRNA-pE194-F"
     primer_bind     complement(4658..4680)
                     /label=B616
                     /note="ptet-R"
     primer_bind     complement(4658..4680)
                     /label=B570
                     /note="rep5m_sp-R"
     primer_bind     complement(4658..4680)
                     /label=B561
                     /note="sgRNA-kansp-R"
     primer_bind     complement(4659..4682)
                     /label=B567
                     /note="(early_lytic)-ptet-R"
     misc_feature    complement(4659..4666)
                     /label=rep
     primer_bind     complement(4667..4690)
                     /label=B560
                     /note="(early)-ptet-R"
     primer_bind     complement(4667..4690)
                     /label=B546
                     /note="(antiR)-ptet-R"
     misc_feature    4667..4672
                     /label=-10
     misc_feature    4677
                     /label=+1
     misc_feature    4683..4701
                     /label=tetO
     primer_bind     complement(4693..4723)
                     /label=LC1090
                     /note="pLC140_RBS_R"
     primer_bind     complement(4699..4738)
                     /label=F139
     primer_bind     complement(4700..4723)
                     /label=B535 - (ANT)tetR-R
                     /note="from pKL55_iTet_RC12"
     primer_bind     4708..4748
                     /label=F141
     primer_bind     4713..4743
                     /label=D010
                     /note="dcas9 F"
     primer_bind     complement(4713..4731)
                     /label=AB2 (2)
                     /note="(ku)ptet-R"
     primer_bind     complement(4713..4731)
                     /label=AB2
                     /note="(ku)ptet-R"
     primer_bind     complement(4713..4723)
                     /label=AB164
                     /note="pAB29v2_bb_R"
     primer_bind     complement(4713..4723)
                     /label=B692
                     /note="(leader2)pWJ153-R"
     primer_bind     complement(4713..4723)
                     /label=B688
                     /note="(CRISPR)pWJ153-F"
     primer_bind     complement(4713..4723)
                     /label=B681
                     /note="(rep)pWJ153-R"
     primer_bind     complement(4714..4739)
                     /label=LC283
                     /note="pTetn_R2"
     misc_feature    4714..4718
                     /label=RBS
     primer_bind     4723..4758
                     /label=LC284
                     /note="pTetn_F2"
     misc_feature    4724..4732
                     /label=RBS
     primer_bind     complement(4725..4749)
                     /label=B544
                     /note="delta-cas9-R"
     primer_bind     complement(4726..4746)
                     /label=LC521
                     /note="pCas9sg_R1"
     CDS             4735..8841
                     /codon_start=1
                     /label=cas9
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     4735..4759
                     /label=B246
                     /note="PyoCas9-LIC-F"
     primer_bind     4735..4759
                     /label=LC673
                     /note="pLC80BB_F"
     primer_bind     4735..4758
                     /label=LC856
                     /note="Cas9_F2"
     primer_bind     4738..4762
                     /label=B446
                     /note="(5'rpoz)-3'cas9-F"
     primer_bind     complement(4742..4760)
                     /label=LC548
                     /note="pCas9sg_R2"
     primer_bind     4743..4782
                     /label=B338
     primer_bind     complement(4743..4782)
                     /label=B337 Cas9D10A-R
                     /note="from JEN25"
     primer_bind     complement(5117..5136)
                     /label=D040
                     /note="GG cas9 R"
     primer_bind     5131..5150
                     /label=D039
                     /note="GG cas9 F"
     primer_bind     5165..5188
                     /label=D005
                     /note="m001 F"
     primer_bind     complement(5195..5214)
                     /label=D006
                     /note="m002 R"
     primer_bind     5363..5384
                     /label=L428
     primer_bind     complement(5753..5772)
                     /label=LC539
                     /note="Cas9seq_2"
     primer_bind     5809..5828
                     /label=B162
                     /note="cas9III-2F"
     primer_bind     6084..6104
                     /label=L429
     primer_bind     6289..6312
                     /label=LC1038
                     /note="BM9_V527F_F"
     primer_bind     complement(6317..6338)
                     /label=LC540
                     /note="Cas9seq_3"
     primer_bind     6783..6803
                     /label=LC1035
                     /note="pLC115_dCas9"
     primer_bind     complement(6819..6839)
                     /label=LC541
                     /note="Cas9seq_4"
     primer_bind     6877..6887
                     /label=LC564
                     /note="pLC61F2"
     primer_bind     6877..6887
                     /label=LC560
                     /note="pLC60F2"
     primer_bind     complement(6991..7003)
                     /label=BG46
                     /note="sbcB_sgRNA_R"
     primer_bind     7234..7273
                     /label=B340
     primer_bind     complement(7234..7273)
                     /label=B339 Cas9H840A
                     /note="from JEN25"
     primer_bind     complement(7321..7341)
                     /label=LC542
                     /note="Cas9seq_5"
     primer_bind     complement(7324..7354)
                     /label=L410
     primer_bind     7324..7354
                     /label=L409
     primer_bind     complement(7485..7495)
                     /label=oGR253
                     /note="phi38.2_rep_F"
     primer_bind     complement(7851..7872)
                     /label=LC543
                     /note="Cas9seq_6"
     primer_bind     8147..8170
                     /label=B221
                     /note="EcoRI-'cas9-F"
     primer_bind     8149..8170
                     /label=L430
     primer_bind     complement(8374..8394)
                     /label=LC544
                     /note="Cas9seq_7"
     primer_bind     8635..8656
                     /label=D012
                     /note="trRNA F"
     primer_bind     complement(8677..8696)
                     /label=D011
                     /note="dcas9 R"
     primer_bind     complement(8707..8760)
                     /label=LC1085
                     /note="R1335A_R"
     primer_bind     complement(8711..8754)
                     /label=LC1083
                     /note="R1333A_R"
     primer_bind     8713..8754
                     /label=LC1082
                     /note="R1333A_F"
     primer_bind     8719..8764
                     /label=LC1084
                     /note="R1335A_F"
     primer_bind     8795..8816
                     /label=LC547
     primer_bind     8813..8834
                     /label=L431
     primer_bind     complement(8815..8838)
                     /label=B441
                     /note="(5'rpoZ)-3'cas9-R"
     primer_bind     complement(8818..8842)
                     /label=F142
     primer_bind     complement(8818..8841)
                     /label=B295
                     /note="cas9-R"
     primer_bind     complement(8818..8838)
                     /label=LC1070
                     /note="pLC128_F1"
     primer_bind     complement(8818..8838)
                     /label=LC1072
                     /note="pLC132_Rev"
     primer_bind     complement(8819..8839)
                     /label=LC1058
                     /note="pLC124/5_F"
     primer_bind     complement(8826..8841)
                     /label=LC314
                     /note="Cas9nbb_R"
     primer_bind     8826..8841
                     /label=BG70
                     /note="psgRNAcos_backbone_R"
     primer_bind     complement(8826..8841)
                     /label=LC519
                     /note="Cas9_R1"
     primer_bind     complement(8826..8841)
                     /label=LC319
                     /note="sgRNA_R2"
     primer_bind     complement(8826..8841)
                     /label=LC343
                     /note="pCasBB_R"
     terminator      8850..8921
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8937..8964
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(8950..8970)
                     /label=dCas9-SmaI-R
     misc_feature    join(8971..9131,1..19)
                     /label=Promoter PpflB
     primer_bind     8971..8990
                     /label=PpflB-SmaI-F
     primer_bind     complement(join(9120..9131,1..34))
                     /label=F237
     primer_bind     complement(join(9131,1..19))
                     /label=PpflB-R
ORIGIN
        1 acaacaaaca tgctacaatt agttgagacc agtctaggtc tcggttttag agctagaaat
       61 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      121 ttttttggag atctgtccat acccatggat tcttcgtctg tttctactcc atcagtctga
      181 cgaccaagag agccataaac accaatagcc ttaacatcat ccccatattt atccaatatt
      241 cgttccttaa tttcatgaac aatctgatat caagcttatc gataccgtcg acgccggcca
      301 gcctcgcaga gcaggattcc cgttgagcac cgccaggtgc gaataaggga cagtgaagaa
      361 ggaacacccg ctcgcgggtg ggcctacttc acctatcctg cccggctgac gccgttggat
      421 acaccaagga aagtctacac gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga
      481 tggatatacc gaaaaaatcg ctataatgac cccgaagcag ggttatgcag cggaaaagcg
      541 ctggtaccca attcgcccta tagtgagtct tcttctctag tcattattat tggtccattc
      601 actattctca ttcccttttc agataatttt agatttgctt ttctaaataa gaatatttgg
      661 agagcaccgt tcttattcag ctattaataa ctcgtcttcc taagcatcct tcaatccttt
      721 taataacaat tatagcatct aatcttcaac aaactggccc gtttgttgaa ctactcttta
      781 ataaaataat ttttccgttc ccaattccac attgcaataa tagaaaatcc atcttcatcg
      841 gctttttcgt catcatctgt atgaatcaaa tcgccttctt ctgtgtcatc aaggtttaat
      901 tttttatgta tttcttttaa caaaccacca taggagatta accttttacg gtgtaaacct
      961 tcctccaaat cagacaaacg tttcaaattc ttttcttcat catcggtcat aaaatccgta
     1021 tcctttacag gatattttgc agtttcgtca attgccgatt gtatatccga tttatattta
     1081 tttttcggtc gattttttat taaaacgtct caaaatcgtt tctgagacgt tttagcgttt
     1141 atttcgttta gttatcggca taatcgttaa aacaggcgtt atcgtagcgt aaaagccctt
     1201 gagcgtagcg tggctttgca gcgaagatgt tgtctgttag attatgaaag ccgatgactg
     1261 aatgaaataa taagcgcagc gtccttctat ttcggttgga ggaggctcaa gggagtttga
     1321 gggaatgaaa ttccctcatg ggtttgattt taaaaattgc ttgcaatttt gccgagcggt
     1381 agcgctggaa aatttttgaa aaaaatttgg aatttggaaa aaaatggggg gaaaggaagc
     1441 gaattttgct tccgtactac gaccccccat taagtgccga gtgccaattt ttgtgccaaa
     1501 aacgctctat cccaactggc tcaagggttt gaggggtttt tcaatcgcca acgaatcgcc
     1561 aacgttttcg ccaacgtttt ttataaatct atatttaagt agctttattg ttgtttttat
     1621 gattacaaag tgatacacta attttataaa attatttgat tggagttttt taaatggtga
     1681 tttcagaatc gaaaaaaaga gttatgattt ctctgacaaa agagcaagat aaaaaattaa
     1741 cagatatggc gaaacaaaaa ggtttttcaa aatctgcggt tgcggcgtta gctatagaag
     1801 aatatgcaag aaaggaatca gaacaaaaaa aataagcgaa agctcgcgtt tttagaagga
     1861 tacgagtttt cgctacttgt ttttgataag gtaattatat catggctatt aaaaatacta
     1921 aagctagaaa ttttggattt ttattatatc ctgactcaat tcctaatgat tggaaagaaa
     1981 aattagagag tttgggcgta tctatggctg tcagtccttt acacgatatg gacgaaaaaa
     2041 aagataaaga tacatggaat agtagtgatg ttatacgaaa tggaaagcac tataaaaaac
     2101 cacactatca cgttatatat attgcacgaa atcctgtaac aatagaaagc gttaggaaca
     2161 agattaagcg aaaattgggg aatagttcag ttgctcatgt tgagatactt gattatatca
     2221 aaggttcata tgaatatttg actcatgaat caaaggacgc tattgctaag aataaacata
     2281 tatacgacaa aaaagatatt ttgaacatta atgattttga tattgaccgc tatataacac
     2341 ttgatgaaag ccaaaaaaga aaattgaaga atttactttt agatatagtg gatgactata
     2401 atttggtaaa tacaaaagat ttaatggctt ttattcgcct taggggagcg gagtttggaa
     2461 ttttaaatac gaatgatgta aaagatattg tttcaacaaa ctctagcgcc tttagattat
     2521 ggtttgaggg caattatcag tgtggatata gagcaagtta tgcaaaggtt cttgatgctg
     2581 aaacggggga aataaaatga caaacaaaga aaaagagtta tttgctgaaa atgaggaatt
     2641 aaaaaaagaa attaaggact taaaagagcg tattgaaaga tacagagaaa tggaagttga
     2701 attaagtaca acaatagatt tattgagagg agggattatt gaataaataa aagcccccct
     2761 gacgaaagtc caagggttta ttgttttcta aaatctgatt accaattaga atgaatattt
     2821 cccaaatatt aaataataaa acaaaaaaat taaaaaagtg tttccaccat tttttcaatt
     2881 tttttataat ttttttaatc tgttatttaa atagtttata gttaaattta cattttcatt
     2941 agtccattca atattctctc caagataact acgaactgct aacaaaattc tctccctatg
     3001 ttctaatgga gaagattcag ccactgcatt tcccgcaata tcttttggta tgattttacc
     3061 cgtgtccata gttaaaatca tacggcataa agttaatata gagttggttt catcatcctg
     3121 ataattatct attaattcct ctgacgaatc cataatggct cttctcacat cagaaaatgg
     3181 aatatcaggt agtaattcct ctaagtcata atttccgtat attcttttat tttttcgttt
     3241 tgcttggtaa agcattatgg ttaaatctga atttaattcc ttctgaggaa tgtatccttg
     3301 ttcataaagc tcttgtaacc attctccata aataaattct tgtttgggag gatgattcca
     3361 cggtaccatt tcttgctgaa taataattgt taattcaata tatcgtaagt tgcttttatc
     3421 tcctattttt tttgaaatag gtctaatttt ttgtataagt atttctttac tttgatctgt
     3481 caatggttca gatacgacga ctaaaaagtc aagatcacta tttggtttta gtccactctc
     3541 aactcctgat ccaaacatgt aagtaccaat aaggttattt tttaaatgtt tccgaagtat
     3601 tttttcactt tattaatttg ttcgtatgta ttcaaatata tcctcctcac tattttgatt
     3661 agtacctatt ttatatccat agttgttaat taaataaact taatttagtt ttatttatag
     3721 atttcattgg cttctaaatt ttttatctag ataataatta ttttagttaa ttttattcta
     3781 gattatatat gatatgatct ttcatttcca taaaactaaa gtaagtgtaa acctattcat
     3841 tgttttaaaa atatctcttg ccagtcacgt tacgttatta gttagttacc atcacggaaa
     3901 aaggttatgc tgcttttaag acccactttc acatttaagt tgtttttcta atccgcatat
     3961 gatcaattca aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat
     4021 agcttgtcgt aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc
     4081 gacttgatgc tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag
     4141 tgcatataat gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc
     4201 gagagtttca tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg
     4261 atgacttagt aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct
     4321 ttccccttct aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc
     4381 gtcgagcaaa gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag
     4441 cttctgggcg agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa
     4501 tgcgctgtta atcactttac ttttatctaa tctagacatc attaattcct cctttttgtt
     4561 gacattatat cattgataga gttatttgtc aaactagttt tttatttgga tcccctcgag
     4621 ttcatgaaaa actaaaaaaa atattgacac tctatcattg atagagcata attaaaataa
     4681 gctctctatc attgatagag tgatatcgaa ttcggaggca tataggaggc aaaaatggat
     4741 aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc ggtgatcact
     4801 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
     4861 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
     4921 cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
     4981 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
     5041 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
     5101 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
     5161 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
     5221 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
     5281 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
     5341 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
     5401 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
     5461 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
     5521 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
     5581 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
     5641 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
     5701 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
     5761 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
     5821 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
     5881 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
     5941 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
     6001 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
     6061 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
     6121 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
     6181 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
     6241 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
     6301 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
     6361 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
     6421 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
     6481 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
     6541 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
     6601 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
     6661 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
     6721 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
     6781 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
     6841 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
     6901 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
     6961 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
     7021 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
     7081 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
     7141 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
     7201 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tcacattgtt
     7261 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
     7321 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
     7381 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
     7441 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
     7501 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
     7561 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
     7621 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
     7681 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
     7741 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
     7801 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
     7861 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
     7921 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
     7981 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
     8041 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
     8101 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
     8161 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
     8221 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
     8281 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
     8341 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
     8401 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
     8461 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
     8521 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
     8581 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
     8641 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
     8701 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
     8761 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
     8821 agtcagctag gaggtgactg accaggcatc aaataaaacg aaaggctcag tcgaaagact
     8881 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct
     8941 caccttcggg tgggcctttc tgcgtttata atatactcct aaattaactt ttaaagcaat
     9001 gaaaatagtg aacattataa ctgttgtgta acagaatgca attagcatat tactgttaca
     9061 caaattagta cagtttctat gttttgacat acatttgatg aaaattgtac ataatttatg
     9121 tgaaaaaaat c
//
