LOCUS       Exported                6884 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector ds-spcas-amp, complete sequence.
ACCESSION   MH015238
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Precise Cas9 targeting enables genomic mutation prevention
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. (2018) In press
  PUBMED    29555762
REFERENCE   2  (bases 1 to 6884)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 
            Blackfan Circle, Floor 5, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 6884)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6884
                     /organism="Cloning vector ds-spcas-amp"
                     /mol_type="other DNA"
                     /db_xref="taxon:2138186"
     regulatory      complement(153..180)
                     /regulatory_class="terminator"
                     /note="rrnB T2 terminator"
     terminator      153..180
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      205..364
                     /regulatory_class="promoter"
                     /note="proC promoter"
     CDS             377..4483
                     /codon_start=1
                     /product="Cas9"
                     /label=Cas9
                     /note="SP-Cas9; derived from Streptococcus pyogenes"
                     /protein_id="AVR59329.1"
                     /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"
     precursor_RNA   4548..4636
                     /product="tracrRNA precursor"
     misc_RNA        4551..4629
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     regulatory      complement(4667..4708)
                     /regulatory_class="terminator"
                     /note="derived from Streptococcus pyogenes; tracrRNA 
                     terminator"
     regulatory      4765..4829
                     /regulatory_class="promoter"
                     /note="J23100 promoter"
     repeat_region   4845..4880
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     rep_origin      5046..5841
                     /label=CloDF13 ori
                     /note="CloDF13 ori"
     rep_origin      5062..5800
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     regulatory      5066..5088
                     /regulatory_class="terminator"
                     /note="putative stem-loop structure; putative cex gene 
                     transcription terminator"
     repeat_region   5066..5073
                     /label=inverted repeat B
                     /note="inverted repeat B"
     regulatory      5077..5082
                     /regulatory_class="minus_35_signal"
                     /note="RNA II"
     repeat_region   5081..5088
                     /label=inverted repeat B
                     /note="inverted repeat B"
     regulatory      5100..5105
                     /regulatory_class="minus_10_signal"
                     /note="RNA II"
     regulatory      5124..5145
                     /regulatory_class="terminator"
                     /note="putative stem-loop structure; putative transcription
                     terminator; RNA I"
     repeat_region   5124..5132
                     /label=inverted repeat A
                     /note="inverted repeat A"
     repeat_region   5137..5145
                     /label=inverted repeat A
                     /note="inverted repeat A"
     regulatory      complement(5228..5233)
                     /regulatory_class="minus_10_signal"
                     /note="RNA I"
     regulatory      complement(5252..5257)
                     /regulatory_class="minus_35_signal"
                     /note="RNA I"
     misc_feature    5708
                     /label=origin of vegetative replication
                     /note="origin of vegetative replication"
     promoter        5848..5939
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5870..5898
                     /regulatory_class="promoter"
                     /note="bla promoter"
     CDS             5949..6809
                     /codon_start=1
                     /gene="bla"
                     /product="Bla"
                     /label=bla
                     /protein_id="AVR59330.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            5949..6809
                     /gene="bla"
                     /label=bla