LOCUS       sequence-311754-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible Cas9HF1 with D1135E mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  AUTHORS   Moreb EA, Hutmacher M, Lynch MD
  TITLE     CRISPR-Cas ""Non-Target"" Sites Inhibit On-Target Cutting Rates.
  JOURNAL   CRISPR J. 2020 Dec;3(6):550-561. doi: 10.1089/crispr.2020.0065.
  PUBMED    33346713
REFERENCE   2  (bases 1 to 6770)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-12"; 
            volume: "3"; issue: "6"; pages: "550-561"
COMMENT     Sequence Label: pEM-Cas9HF1-D1135E
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..633)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVL*DQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(646..680)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        681..736
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    717..735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(772..2259,2260..2262,2263..2751,2752..2754,2755..2853,
                     2854..2856,2857..3546,3547..3549,3550..4875)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF2
                     /note="carries the mutations N497A, R661A, Q695A, Q926A, 
                     and D1135E"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             772..4875
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4876..4908
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag
                     /note="wild-type LAA variant"
                     /translation="AANDENYALAA"
     terminator      4935..5006
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5022..5049
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5077..5094)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5211..5756)
                     /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(5291..5310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5870..5964)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5985..6644)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6559..6578
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6645..6747)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tacaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taattgctag cattgtacct
      661 aggactgagc tagccataaa gttgacactc tatcgttgat agagttattt taccactccc
      721 tatcagtgat agagaaaaga attcaaaaga tctaaagagg acttcggatc tatggataag
      781 aaatactcaa taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat
      841 gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc
      901 aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgga agcgactcgc
      961 ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttg ttatctacag
     1021 gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag
     1081 tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta
     1141 gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta
     1201 gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag
     1261 tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa
     1321 ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca
     1381 agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa
     1441 aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct
     1501 ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa
     1561 ttacagcttt caaaagatac ttacgatgat gatttagata atttattggc gcaaattgga
     1621 gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca
     1681 gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa
     1741 cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt
     1801 ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt
     1861 gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg
     1921 gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg
     1981 acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg
     2041 agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc
     2101 ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca
     2161 tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat
     2221 aaaggtgctt cagctcaatc atttattgaa cgcatgacag cgtttgataa aaatcttcca
     2281 aatgaaaaag tactaccaaa acatagtttg ctttatgagt attttacggt ttataacgaa
     2341 ttgacaaagg tcaaatatgt tactgaagga atgcgaaaac cagcatttct ttcaggtgaa
     2401 cagaagaaag ccattgttga tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa
     2461 ttaaaagaag attatttcaa aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt
     2521 gaagatagat ttaatgcttc attaggtacc taccatgatt tgctaaaaat tattaaagat
     2581 aaagattttt tggataatga agaaaatgaa gatatcttag aggatattgt tttaacattg
     2641 accttatttg aagataggga gatgattgag gaaagactta aaacatatgc tcacctcttt
     2701 gatgataagg tgatgaaaca gcttaaacgt cgccgttata ctggttgggg agcgttgtct
     2761 cgaaaattga ttaatggtat tagggataag caatctggca aaacaatatt agattttttg
     2821 aaatcagatg gttttgccaa tcgcaatttt atggcgctga tccatgatga tagtttgaca
     2881 tttaaagaag acattcaaaa agcacaagtg tctggacaag gcgatagttt acatgaacat
     2941 attgcaaatt tagctggtag ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt
     3001 gttgatgaat tggtcaaagt aatggggcgg cataagccag aaaatatcgt tattgaaatg
     3061 gcacgtgaaa atcagacaac tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga
     3121 atcgaagaag gtatcaaaga attaggaagt cagattctta aagagcatcc tgttgaaaat
     3181 actcaattgc aaaatgaaaa gctctatctc tattatctcc aaaatggaag agacatgtat
     3241 gtggaccaag aattagatat taatcgttta agtgattatg atgtcgatca cattgttcca
     3301 caaagtttcc ttaaagacga ttcaatagac aataaggtct taacgcgttc tgataaaaat
     3361 cgtggtaaat cggataacgt tccaagtgaa gaagtagtca aaaagatgaa aaactattgg
     3421 agacaacttc taaacgccaa gttaatcact caacgtaagt ttgataattt aacgaaagct
     3481 gaacgtggag gtttgagtga acttgataaa gctggtttta tcaaacgcca attggttgaa
     3541 actcgcgcga tcactaagca tgtggcacaa attttggata gtcgcatgaa tactaaatac
     3601 gatgaaaatg ataaacttat tcgagaggtt aaagtgatta ccttaaaatc taaattagtt
     3661 tctgacttcc gaaaagattt ccaattctat aaagtacgtg agattaacaa ttaccatcat
     3721 gcccatgatg cgtatctaaa tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa
     3781 cttgaatcgg agtttgtcta tggtgattat aaagtttatg atgttcgtaa aatgattgct
     3841 aagtctgagc aagaaatagg caaagcaacc gcaaaatatt tcttttactc taatatcatg
     3901 aacttcttca aaacagaaat tacacttgca aatggagaga ttcgcaaacg ccctctaatc
     3961 gaaactaatg gggaaactgg agaaattgtc tgggataaag ggcgagattt tgccacagtg
     4021 cgcaaagtat tgtccatgcc ccaagtcaat attgtcaaga aaacagaagt acagacaggc
     4081 ggattctcca aggagtcaat tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa
     4141 aaagactggg atccaaaaaa atatggtggt tttgaaagtc caacggtagc ttattcagtc
     4201 ctagtggttg ctaaggtgga aaaagggaaa tcgaagaagt taaaatccgt taaagagtta
     4261 ctagggatca caattatgga aagaagttcc tttgaaaaaa atccgattga ctttttagaa
     4321 gctaaaggat ataaggaagt taaaaaagac ttaatcatta aactacctaa atatagtctt
     4381 tttgagttag aaaacggtcg taaacggatg ctggctagtg ccggagaatt acaaaaagga
     4441 aatgagctgg ctctgccaag caaatatgtg aattttttat atttagctag tcattatgaa
     4501 aagttgaagg gtagtccaga agataacgaa caaaaacaat tgtttgtgga gcagcataag
     4561 cattatttag atgagattat tgagcaaatc agtgaatttt ctaagcgtgt tattttagca
     4621 gatgccaatt tagataaagt tcttagtgca tataacaaac atagagacaa accaatacgt
     4681 gaacaagcag aaaatattat tcatttattt acgttgacga atcttggagc tcccgctgct
     4741 tttaaatatt ttgatacaac aattgatcgt aaacgatata cgtctacaaa agaagtttta
     4801 gatgccactc ttatccatca atccatcact ggtctttatg aaacacgcat tgatttgagt
     4861 cagctaggag gtgacgctgc taacgacgaa aactacgctc tggctgctta actcgagtaa
     4921 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4981 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     5041 ctttctgcgt ttatacctag ggatatattc cgcttcctcg ctcactgact cgctacgctc
     5101 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga
     5161 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg
     5221 ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg
     5281 acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt
     5341 cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg
     5401 cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc
     5461 cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag
     5521 acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga
     5581 agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa
     5641 gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca
     5701 aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag
     5761 atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg
     5821 tagcacctga agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa
     5881 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca
     5941 ttactggatc tatcaacagg agtccaagcg agctcgatat caaattacgc cccgccctgc
     6001 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     6061 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     6121 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     6181 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     6241 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     6301 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     6361 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     6421 atacgaaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     6481 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     6541 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     6601 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     6661 cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga
     6721 aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccagatatc
//
