LOCUS       Exported                8068 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression plasmid for Sp-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    MSP712
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8068)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 8068)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8068
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /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)."
     CDS             71..4174
                     /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
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4181..4201
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4208..4273
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        4343..4361
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        4430..4505
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4507..4525)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4521..4568
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4789..5448)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5363..5382
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5449..5551)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5943..5960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6077..6621)
                     /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(6157..6176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6846..7928)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7890..7909
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7929..8006)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        8052..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggataaaa agtattctat tggtttagcc atcggcacta attccgttgg
      121 atgggctgtc ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa
      181 cacagaccgt cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga
      241 aacggcagag gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa
      301 ccgaatatgt tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt
      361 ctttcaccgt ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc
      421 catctttgga aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca
      481 cctcagaaaa aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc
      541 tcttgcccat atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga
      601 caactcggat gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga
      661 agagaaccct ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc
      721 taaatcccga cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt
      781 gttcggtaac cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga
      841 cttagctgaa gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa
      901 tctactggca caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag
      961 cgatgcaatc ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt
     1021 atccgcttca atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc
     1081 cctagtccgt cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa
     1141 cgggtacgca ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa
     1201 acccatatta gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga
     1261 tctactgcga aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg
     1321 cgaattgcat gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg
     1381 tgaaaagatt gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg
     1441 agggaactct cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccctggaa
     1501 ttttgaggaa gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa
     1561 ctttgacaag aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta
     1621 tttcacagtg tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc
     1681 cgcctttcta agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg
     1741 caaagtgaca gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc
     1801 tgtcgagatc tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct
     1861 cctaaagata attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga
     1921 agatatagtg ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa
     1981 aacatacgct cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac
     2041 gggctgggga cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa
     2101 aactattctc gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat
     2161 ccatgatgac tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg
     2221 ggactcattg cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat
     2281 actccagaca gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga
     2341 aaacattgta atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag
     2401 tcgagagcgg atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa
     2461 ggagcatcct gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca
     2521 aaatggaagg gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga
     2581 cgtcgatgcc attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct
     2641 tacacgctcg gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa
     2701 gaaaatgaag aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt
     2761 cgataactta actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat
     2821 taaacgtcag ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc
     2881 ccgaatgaat acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac
     2941 tttaaagtca aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga
     3001 gataaataac taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact
     3061 cattaagaaa tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga
     3121 cgtccgtaag atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt
     3181 cttttattct aacattatga atttctttaa gacggaaatc actctggcaa acggagagat
     3241 acgcaaacga cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg
     3301 ccgggacttc gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa
     3361 aactgaggtg cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga
     3421 taagctcatc gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc
     3481 tacagttgcc tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact
     3541 gaagtcagtc aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa
     3601 ccccatcgac ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa
     3661 actaccaaag tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc
     3721 cggagagctt caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta
     3781 tttagcgtcc cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact
     3841 ttttgttgag cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag
     3901 taagagagtc atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca
     3961 cagggataaa cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa
     4021 cctcggcgct ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac
     4081 ttctaccaag gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga
     4141 aactcggata gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt
     4201 ctcgagcgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     4261 tgacgatgac aagtgaagcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
     4321 tacacggccg cataatcgaa attaatacga ctcactatag ggagacccat gccatagcgt
     4381 tgttcggaat atgaattttt gaacagattc accaacacct agtggtctcg ttttagagct
     4441 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     4501 ggtgctccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     4561 gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
     4621 ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga
     4681 cgaccgggtc gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca
     4741 ggcgtagcaa ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc
     4801 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     4861 acagacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     4921 atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     4981 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
     5041 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     5101 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     5161 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     5221 tgccatacgg aactccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     5281 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     5341 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     5401 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
     5461 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     5521 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca
     5581 gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt
     5641 cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg
     5701 tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt
     5761 cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga
     5821 gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc
     5881 aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct
     5941 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     6001 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     6061 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     6121 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttcccctgg
     6181 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
     6241 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca
     6301 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact
     6361 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta
     6421 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca
     6481 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
     6541 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg
     6601 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt
     6661 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     6721 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6781 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6841 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6961 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     7021 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     7081 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     7141 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     7201 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     7261 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     7321 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     7381 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     7441 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     7501 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     7561 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     7621 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     7681 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     7741 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     7801 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     7861 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     7921 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     7981 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     8041 cattaggaaa ttaatacgac tcactata
//
