LOCUS       Exported                6182 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid constitutively expressing eSpdCas9 protein.
ACCESSION   .
VERSION     .
KEYWORDS    peSpdCas9-J23109
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  AUTHORS   Guo J, Wang T, Guan C, Liu B, Luo C, Xie Z, Zhang C, Xing XH
  TITLE     Improved sgRNA design in bacteria via genome-wide activity 
            profiling.
  JOURNAL   Nucleic Acids Res. 2018 Aug 21;46(14):7052-7069. doi: 
            10.1093/nar/gky572.
  PUBMED    29982721
REFERENCE   2  (bases 1 to 6182)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6182
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..4107
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEALY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4131..4202
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4218..4245
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4273..4290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4407..4952)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4487..4506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5066..5160
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5184..5999)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5907..5926
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5991..6010
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     RBS             6165..6176
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg
       61 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
      121 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
      181 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
      241 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
      301 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
      361 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
      421 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
      481 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
      541 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
      601 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
      661 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
      721 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
      781 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
      841 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
      901 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
      961 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
     1021 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
     1081 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
     1141 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
     1201 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
     1261 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
     1321 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
     1381 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
     1441 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
     1501 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
     1561 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
     1621 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
     1681 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
     1741 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
     1801 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
     1861 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
     1921 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
     1981 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
     2041 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
     2101 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
     2161 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
     2221 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
     2281 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
     2341 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
     2401 gttgaaaata ctcaattgca aaatgaagca ctctatctct attatctcca aaatggaaga
     2461 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc
     2521 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
     2581 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
     2641 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
     2701 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
     2761 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
     2821 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
     2881 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
     2941 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
     3001 tatccagcac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
     3061 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
     3121 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaagca
     3181 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
     3241 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
     3301 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
     3361 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
     3421 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
     3481 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
     3541 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
     3601 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
     3661 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
     3721 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
     3781 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
     3841 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
     3901 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
     3961 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
     4021 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
     4081 gatttgagtc agctaggagg tgactaactc gagtaaggat ctccaggcat caaataaaac
     4141 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     4201 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acctagggat
     4261 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     4321 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     4381 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     4441 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     4501 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     4561 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     4621 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     4681 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     4741 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     4801 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt
     4861 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     4921 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     4981 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     5041 gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga
     5101 gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc
     5161 caagcgagct cgatatcaaa ttattagaaa aactcatcga gcatcaaatg aaactgcaat
     5221 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5281 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5341 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5401 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     5461 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5521 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     5581 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5641 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     5701 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     5761 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     5821 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     5881 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     5941 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata
     6001 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     6061 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc cctgcagttt
     6121 acagctagct cagtcctagg gactgtgcta gcggccgcag atctaaagag gagaaaggat
     6181 ct
//
