LOCUS       sequence-201611-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified from pCas9-CR4 (Addgene: 62655) to remove the ssrA tag from
            Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar
            R, Lynch MD
  TITLE     Managing the SOS Response for Enhanced CRISPR-Cas-Based 
            Recombineering in E. coli through Transient Inhibition of Host RecA 
            Activity.
  JOURNAL   ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174.
  PUBMED    28915012
REFERENCE   2  (bases 1 to 6737)
  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: "ACS Synth 
            Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174."
COMMENT     Sequence Label: pEM-Cas9noSsrA
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..160
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(188..215)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(231..302)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(326..4432)
                     /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             complement(329..4432)
                     /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"
     promoter        4468..4523
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4469..4487
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        4524..4558
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     CDS             4571..5197
                     /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     complement(4647..4666)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5227..5329
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             5330..5989
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5396..5415)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6010..6104
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(6218..6737,1..26)
                     /direction=RIGHT
                     /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     6664..6683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt
       61 aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg
      121 ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatccctag
      181 gtataaacgc agaaaggccc acccgaaggt gagccagtgt gactctagta gagagcgttc
      241 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
      301 tgatgcctgg agatccttac tcgagttagt cacctcctag ctgactcaaa tcaatgcgtg
      361 tttcataaag accagtgatg gattgatgga taagagtggc atctaaaact tcttttgtag
      421 acgtatatcg tttacgatca attgttgtat caaaatattt aaaagcagcg ggagctccaa
      481 gattcgtcaa cgtaaataaa tgaataatat tttctgcttg ttcacgtatt ggtttgtctc
      541 tatgtttgtt atatgcacta agaactttat ctaaattggc atctgctaaa ataacacgct
      601 tagaaaattc actgatttgc tcaataatct catctaaata atgcttatgc tgctccacaa
      661 acaattgttt ttgttcgtta tcttctggac tacccttcaa cttttcataa tgactagcta
      721 aatataaaaa attcacatat ttgcttggca gagccagctc atttcctttt tgtaattctc
      781 cggcactagc cagcatccgt ttacgaccgt tttctaactc aaaaagacta tatttaggta
      841 gtttaatgat taagtctttt ttaacttcct tatatccttt agcttctaaa aagtcaatcg
      901 gatttttttc aaaggaactt ctttccataa ttgtgatccc tagtaactct ttaacggatt
      961 ttaacttctt cgatttccct ttttccacct tagcaaccac taggactgaa taagctaccg
     1021 ttggactatc aaaaccacca tatttttttg gatcccagtc ttttttacga gcaataagct
     1081 tgtccgaatt tctttttggt aaaattgact ccttggagaa tccgcctgtc tgtacttctg
     1141 ttttcttgac aatattgact tggggcatgg acaatacttt gcgcactgtg gcaaaatctc
     1201 gccctttatc ccagacaatt tctccagttt ccccattagt ttcgattaga gggcgtttgc
     1261 gaatctctcc atttgcaagt gtaatttctg ttttgaagaa gttcatgata ttagagtaaa
     1321 agaaatattt tgcggttgct ttgcctattt cttgctcaga cttagcaatc attttacgaa
     1381 catcataaac tttataatca ccatagacaa actccgattc aagttttgga tatttcttaa
     1441 tcaaagcagt tccaacgacg gcatttagat acgcatcatg ggcatgatgg taattgttaa
     1501 tctcacgtac tttatagaat tggaaatctt ttcggaagtc agaaactaat ttagatttta
     1561 aggtaatcac tttaacctct cgaataagtt tatcattttc atcgtattta gtattcatgc
     1621 gactatccaa aatttgtgcc acatgcttag tgatttggcg agtttcaacc aattggcgtt
     1681 tgataaaacc agctttatca agttcactca aacctccacg ttcagctttc gttaaattat
     1741 caaacttacg ttgagtgatt aacttggcgt ttagaagttg tctccaatag tttttcatct
     1801 ttttgactac ttcttcactt ggaacgttat ccgatttacc acgattttta tcagaacgcg
     1861 ttaagacctt attgtctatt gaatcgtctt taaggaaact ttgtggaaca atgtgatcga
     1921 catcataatc acttaaacga ttaatatcta attcttggtc cacatacatg tctcttccat
     1981 tttggagata atagagatag agcttttcat tttgcaattg agtattttca acaggatgct
     2041 ctttaagaat ctgacttcct aattctttga taccttcttc gattcgtttc atacgctctc
     2101 gcgaattttt ctggcccttt tgagttgtct gattttcacg tgccatttca ataacgatat
     2161 tttctggctt atgccgcccc attactttga ccaattcatc aacaactttt acagtctgta
     2221 aaataccttt tttaatagca gggctaccag ctaaatttgc aatatgttca tgtaaactat
     2281 cgccttgtcc agacacttgt gctttttgaa tgtcttcttt aaatgtcaaa ctatcatcat
     2341 ggatcagctg cataaaattg cgattggcaa aaccatctga tttcaaaaaa tctaatattg
     2401 ttttgccaga ttgcttatcc ctaataccat taatcaattt tcgagacaaa cgtccccaac
     2461 cagtataacg gcgacgttta agctgtttca tcaccttatc atcaaagagg tgagcatatg
     2521 ttttaagtct ttcctcaatc atctccctat cttcaaataa ggtcaatgtt aaaacaatat
     2581 cctctaagat atcttcattt tcttcattat ccaaaaaatc tttatcttta ataattttta
     2641 gcaaatcatg gtaggtacct aatgaagcat taaatctatc ttcaactcct gaaatttcaa
     2701 cactatcaaa acattctatt tttttgaaat aatcttcttt taattgctta acggttactt
     2761 ttcgatttgt tttgaagagt aaatcaacaa tggctttctt ctgttcacct gaaagaaatg
     2821 ctggttttcg cattccttca gtaacatatt tgacctttgt caattcgtta taaaccgtaa
     2881 aatactcata aagcaaacta tgttttggta gtactttttc atttggaaga tttttatcaa
     2941 agtttgtcat gcgttcaata aatgattgag ctgaagcacc tttatcgaca acttcttcaa
     3001 aattccatgg ggtaattgtt tcttcagact tccgagtcat ccatgcaaaa cgactattgc
     3061 cacgcgccaa tggaccaaca taataaggaa ttcgaaaagt caagattttt tcaatcttct
     3121 cacgattgtc ttttaaaaat ggataaaagt cttcttgtct tctcaaaata gcatgcagct
     3181 cacccaagtg aatttgatgg ggaatagagc cgttgtcaaa ggtccgttgc ttgcgcagca
     3241 aatcttcacg atttagtttc accaataatt cctcagtacc atccattttt tctaaaattg
     3301 gtttgataaa tttataaaat tcttcttggc tagctccccc atcaatataa cctgcatatc
     3361 cgttttttga ttgatcaaaa aagatttctt tatacttttc tggaagttgt tgtcgaacta
     3421 aagcttttaa aagagtcaag tcttgatgat gttcatcgta gcgtttaatc attgaagctg
     3481 ataggggagc cttagttatt tcagtattta ctcttaggat atctgaaagt aaaatagcat
     3541 ctgataaatt cttagctgcc aaaaacaaat cagcatattg atctccaatt tgcgccaata
     3601 aattatctaa atcatcatcg taagtatctt ttgaaagctg taatttagca tcttctgcca
     3661 aatcaaaatt tgatttaaaa ttaggggtca aacccaatga caaagcaatg agattcccaa
     3721 ataagccatt tttcttctca ccggggagct gagcaatgag attttctaat cgtcttgatt
     3781 tactcaatcg tgcagaaaga atcgctttag catctactcc acttgcgtta atagggtttt
     3841 cttcaaataa ttgattgtag gtttgtacca actggataaa tagtttgtcc acatcactat
     3901 tatcaggatt taaatctccc tcaatcaaaa aatgaccacg aaacttaatc atatgcgcta
     3961 aggccaaata gattaagcgc aaatccgctt tatcagtaga atctaccaat ttttttcgca
     4021 gatgatagat agttggatat ttctcatgat aagcaacttc atctactata tttccaaaaa
     4081 taggatgacg ttcatgcttc ttgtcttctt ccaccaaaaa agactcttca agtcgatgaa
     4141 agaaactatc atctactttc gccatctcat ttgaaaaaat ctcctgtaga taacaaatac
     4201 gattcttccg acgtgtatac cttctacgag ctgtccgttt gagacgagtc gcttccgctg
     4261 tctctccact gtcaaataaa agagccccta taagattttt tttgatactg tggcggtctg
     4321 tatttcccag aaccttgaac tttttagacg gaaccttata ttcatcagtg atcaccgccc
     4381 atccgacgct atttgtgccg atatctaagc ctattgagta tttcttatcc atagatccga
     4441 agtcctcttt agatcttttg aattcttttc tctatcactg atagggagtg gtaaaataac
     4501 tctatcaacg atagagtgtc aactttatgg ctagctcagt cctaggtaca atgctagcaa
     4561 ttaggaatta atgatgtcta gattagataa aagtaaagtg attaacagcg cattagagct
     4621 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     4681 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     4741 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     4801 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     4861 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     4921 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     4981 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     5041 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     5101 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     5161 attagaaaaa caacttaaat gtgaaagtgg gtcttaagac gtcgatatct ggcgaaaatg
     5221 agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
     5281 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     5341 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     5401 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     5461 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     5521 ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg agctggtgat
     5581 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     5641 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     5701 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     5761 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     5821 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     5881 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     5941 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttgatatcga
     6001 gctcgcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     6061 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagtaaca
     6121 acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat tgcactgaaa
     6181 tctagaaata ttttatctga ttaataagat gatcttcttg agatcgtttt ggtctgcgcg
     6241 taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct
     6301 gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg
     6361 tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta
     6421 ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag
     6481 ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg
     6541 gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa
     6601 cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc
     6661 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg
     6721 tcagatttcg tgatgct
//
