LOCUS       sequence-217189-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCas9CR4 with the xCas9 mutations allowing NG PAM sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  TITLE     pCas9CR4 with point mutations from Cas9X for NG PAM sites
  JOURNAL   Unpublished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            "Unpublished"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: pxCas9CR4
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..56
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             92..4195
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIKRMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4196..4228
                     /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      4255..4326
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4342..4369
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4397..4414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4531..5076)
                     /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(4611..4630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5190..5284)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5305..5964)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5879..5898
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5965..6067)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6097..6723)
                     /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
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     6628..6647
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(6736..6770)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
ORIGIN
        1 gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat agagaaaaga
       61 attcaaaaga tctaaagagg acttcggatc tatggataag aaatactcaa taggcttaga
      121 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
      181 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
      241 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
      301 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
      361 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
      421 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
      481 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
      541 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
      601 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
      661 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
      721 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
      781 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
      841 taattttaaa tcaaattttg atttggcaga agatactaaa ttacagcttt caaaagatac
      901 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
      961 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     1021 tgaaataact aaggctcccc tatcagcttc aatgattaaa ctctacgatg aacatcatca
     1081 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     1141 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     1201 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     1261 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggcattat
     1321 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     1381 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     1441 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     1501 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     1561 atttattaaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     1621 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     1681 tactgaagga atgcgaaaac cagcatttct ttcaggtgac cagaagaaag ccattgttga
     1741 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     1801 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     1861 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     1921 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     1981 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     2041 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     2101 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     2161 tcgcaatttt atccagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     2221 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     2281 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     2341 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     2401 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     2461 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     2521 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     2581 taatcgttta agtgattatg atgtcgatca cattgttcca caaagtttcc ttaaagacga
     2641 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     2701 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     2761 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     2821 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     2881 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     2941 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     3001 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     3061 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta
     3121 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     3181 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     3241 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg
     3301 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     3361 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     3421 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     3481 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     3541 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     3601 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     3661 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     3721 taaacggatg ctggctagtg ccggagtatt acaaaaagga aatgagctgg ctctgccaag
     3781 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     3841 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     3901 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     3961 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     4021 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     4081 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     4141 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgacgctgc
     4201 taacgacgaa aactacgctc tggctgctta actcgagtaa ggatctccag gcatcaaata
     4261 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     4321 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag
     4381 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
     4441 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     4501 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     4561 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     4621 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     4681 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     4741 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     4801 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     4861 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     4921 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     4981 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     5041 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata
     5101 aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     5161 atacgatata agttgttact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa
     5221 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
     5281 agtccaagcg agctcgatat caaattacgc cccgccctgc cactcatcgc agtactgttg
     5341 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     5401 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     5461 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     5521 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     5581 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     5641 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     5701 aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt ccggatgagc
     5761 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     5821 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     5881 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     5941 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     6001 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     6061 ccgatcaacg tctcattttc gccagatatc gacgtcttaa gacccacttt cacatttaag
     6121 ttgtttttct aatccgcata tgatcaattc aaggccgaat aagaaggctg gctctgcacc
     6181 ttggtgatca aataattcga tagcttgtcg taataatggc ggcatactat cagtagtagg
     6241 tgtttccctt tcttctttag cgacttgatg ctcttgatct tccaatacgc aacctaaagt
     6301 aaaatgcccc acagcgctga gtgcatataa tgcattctct agtgaaaaac cttgttggca
     6361 taaaaaggct aattgatttt cgagagtttc atactgtttt tctgtaggcc gtgtacctaa
     6421 atgtactttt gctccatcgc gatgacttag taaagcacat ctaaaacttt tagcgttatt
     6481 acgtaaaaaa tcttgccagc tttccccttc taaagggcaa aagtgagtat ggtgcctatc
     6541 taacatctca atggctaagg cgtcgagcaa agcccgctta ttttttacat gccaatacaa
     6601 tgtaggctgc tctacaccta gcttctgggc gagtttacgg gttgttaaac cttcgattcc
     6661 gacctcatta agcagctcta atgcgctgtt aatcacttta cttttatcta atctagacat
     6721 cattaattcc taattgctag cattgtacct aggactgagc tagccataaa
//
