LOCUS       sequence-233288-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCas9CR4 with stop codon to prevent translation of ssrA degradation 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  TITLE     pCas9CR4 variants for increased targeting space
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6765)
  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            "Unpublished"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: pCas9CR5
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             36..4142
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4194..4265
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4281..4308
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4336..4353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4470..5015)
                     /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(4550..4569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5129..5223)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5244..5903)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5818..5837
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5904..6006)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6036..6662)
                     /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     6567..6586
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(6675..6709)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        6710..6765
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6746..6764
                     /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 tggttttgat 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 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc
     4081 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact
     4141 gacgacgaaa actacgctct ggctgcttaa ctcgagtaag gatctccagg catcaaataa
     4201 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4261 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctagg
     4321 gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc
     4381 ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag
     4441 ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat
     4501 cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag
     4561 gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt
     4621 gtcattccgc tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta
     4681 ggcagttcgc tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc
     4741 cttatccggt aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc
     4801 agcagccact ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct
     4861 aaactgaaag gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag
     4921 agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag
     4981 agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa
     5041 aatatttcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
     5101 tacgatataa gttgttacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac
     5161 cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga
     5221 gtccaagcga gctcgatatc aaattacgcc ccgccctgcc actcatcgca gtactgttgt
     5281 aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc
     5341 gccagcggca tcagcacctt gtcgccctgc gtataatatt tgcccatggt gaaaacgggg
     5401 gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga
     5461 ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca
     5521 ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat
     5581 tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga
     5641 acactatccc atatcaccag ctcaccgtct ttcattgcca tacgaaattc cggatgagca
     5701 ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt
     5761 acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca
     5821 actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta
     5881 tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca
     5941 aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc
     6001 cgatcaacgt ctcattttcg ccagatatcg acgtcttaag acccactttc acatttaagt
     6061 tgtttttcta atccgcatat gatcaattca aggccgaata agaaggctgg ctctgcacct
     6121 tggtgatcaa ataattcgat agcttgtcgt aataatggcg gcatactatc agtagtaggt
     6181 gtttcccttt cttctttagc gacttgatgc tcttgatctt ccaatacgca acctaaagta
     6241 aaatgcccca cagcgctgag tgcatataat gcattctcta gtgaaaaacc ttgttggcat
     6301 aaaaaggcta attgattttc gagagtttca tactgttttt ctgtaggccg tgtacctaaa
     6361 tgtacttttg ctccatcgcg atgacttagt aaagcacatc taaaactttt agcgttatta
     6421 cgtaaaaaat cttgccagct ttccccttct aaagggcaaa agtgagtatg gtgcctatct
     6481 aacatctcaa tggctaaggc gtcgagcaaa gcccgcttat tttttacatg ccaatacaat
     6541 gtaggctgct ctacacctag cttctgggcg agtttacggg ttgttaaacc ttcgattccg
     6601 acctcattaa gcagctctaa tgcgctgtta atcactttac ttttatctaa tctagacatc
     6661 attaattcct aattgctagc attgtaccta ggactgagct agccataaag ttgacactct
     6721 atcgttgata gagttatttt accactccct atcagtgata gagaa
//
