LOCUS       sequence-233296-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCas9CR4 with VQR mutations for changing PAM site specificity to 
            NGAN or NGNG from Kleinstiver et al. 2015.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  TITLE     pCas9CR4 variants for increased targeting space
  JOURNAL   Unpublished
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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCas9CR4-VQR
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(36..3437,3438..3440,3441..4037,4038..4040,4041..4043,
                     4044..4046,4047..4139)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             36..4139
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4140..4172
                     /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      4199..4270
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4286..4313
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4341..4358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4475..5020)
                     /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(4555..4574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5134..5228)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5249..5908)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5823..5842
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5909..6011)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6041..6667)
                     /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
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELSDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     6572..6591
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(6680..6714)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        6715..6770
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6751..6769
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aagaattcaa aagatctaaa gaggacttcg gatctatgga taagaaatac tcaataggct
       61 tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
      121 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
      181 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
      241 gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg
      301 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
      361 aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc
      421 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
      481 cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt
      541 tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg
      601 tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta
      661 aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc
      721 tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga
      781 cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag
      841 atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt
      901 tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa
      961 atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc
     1021 atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag
     1081 aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc
     1141 aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat
     1201 tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
     1261 ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact
     1321 tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc
     1381 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt
     1441 ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc
     1501 aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac
     1561 caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat
     1621 atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg
     1681 ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt
     1741 tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg
     1801 cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat tttttggata
     1861 atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata
     1921 gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga
     1981 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
     2041 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
     2101 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
     2161 aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
     2221 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
     2281 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
     2341 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
     2401 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
     2461 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
     2521 atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag
     2581 acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata
     2641 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
     2701 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
     2761 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
     2821 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
     2881 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
     2941 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
     3001 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
     3061 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
     3121 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
     3181 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
     3241 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
     3301 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
     3361 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
     3421 aaaaatatgg tggttttgtt agtccaacgg tagcttattc agtcctagtg gttgctaagg
     3481 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
     3541 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
     3601 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
     3661 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc
     3721 caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc
     3781 cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga
     3841 ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata
     3901 aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata
     3961 ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata
     4021 caacaattga tcgtaaacag tatcggtcta caaaagaagt tttagatgcc actcttatcc
     4081 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgacg
     4141 ctgctaacga cgaaaactac gctctggctg cttaactcga gtaaggatct ccaggcatca
     4201 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     4261 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac
     4321 ctagggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     4381 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     4441 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     4501 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     4561 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     4621 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     4681 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     4741 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     4801 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     4861 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
     4921 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     4981 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
     5041 gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
     5101 ccccatacga tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg
     5161 gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa
     5221 caggagtcca agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact
     5281 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     5341 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     5401 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     5461 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     5521 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     5581 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     5641 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacga aattccggat
     5701 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     5761 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     5821 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     5881 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     5941 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     6001 cgtgccgatc aacgtctcat tttcgccaga tatcgacgtc ttaagaccca ctttcacatt
     6061 taagttgttt ttctaatccg catatgatca attcaaggcc gaataagaag gctggctctg
     6121 caccttggtg atcagataat tcgatagctt gtcgtaataa tggcggcata ctatcagtag
     6181 taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat acgcaaccta
     6241 aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt ctctagtgaa aaaccttgtt
     6301 ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta ggccgtgtac
     6361 ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt
     6421 tattacgtaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga gtatggtgcc
     6481 tatctaacat ctcaatggct aaggcgtcga gcaaagcccg cttatttttt acatgccaat
     6541 acaatgtagg ctgctctaca cctagcttct gggcgagttt acgggttgtt aaaccttcga
     6601 ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta tctaatctag
     6661 acatcattaa ttcctaattg ctagcattgt acctaggact gagctagcca taaagttgac
     6721 actctatcgt tgatagagtt attttaccac tccctatcag tgatagagaa
//
