LOCUS       sequence-229686-        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  JOURNAL   Unpublished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            "Unpublished"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             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
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     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             772..4878
                     /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             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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4930..5001
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5017..5044
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5072..5089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5206..5751)
                     /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(5286..5305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5865..5959)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5980..6639)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6554..6573
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6640..6742)
                     /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 tccaatacgc 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 agcgactcgt
      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 cgcatgacaa actttgataa 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 acgtttgtct
     2761 cgaaaattga ttaatggtat tagggataag caatctggca aaacaatatt agattttttg
     2821 aaatcagatg gttttgccaa tcgcaatttt atgcagctga 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 actcgccaaa 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 tttgatagtc 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 gtgactgacg acgaaaacta cgctctggct gcttaactcg agtaaggatc
     4921 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     4981 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     5041 tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
     5101 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
     5161 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
     5221 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
     5281 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
     5341 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
     5401 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
     5461 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
     5521 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
     5581 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
     5641 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
     5701 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
     5761 cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca
     5821 cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc accaataaaa
     5881 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact
     5941 ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc cctgccactc
     6001 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     6061 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     6121 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     6181 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     6241 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     6301 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     6361 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     6421 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     6481 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     6541 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     6601 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     6661 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
     6721 ggaacctctt acgtgccgat caacgtctca ttttcgccag atatc
//
