LOCUS       sequence-231124-        6178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo golden gate assembly CD part for Cas9 (S. pyogenes 
            gRNA-targeted endonuclease Cas9). Please see Supplemental Documents 
            for annotated Genbank file..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6178)
  TITLE     CIDAR MoClo Extension
REFERENCE   2  (bases 1 to 6178)
  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"
COMMENT     Sequence Label: C113m
FEATURES             Location/Qualifiers
     source          1..6178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     471..490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(709..813)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     881..899
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(903..946)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             969..5075
                     /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             969..5072
                     /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"
     primer_bind     complement(5096..5113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5267..5855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5347..5366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6026..6178,1..639,640..708))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
       61 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
      121 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
      181 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
      241 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
      301 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
      361 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
      421 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
      481 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
      541 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
      601 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
      661 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
      721 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
      781 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
      841 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggca
      901 gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgga attcgcggcc
      961 ggtctcaaat ggataagaaa tactcaatag gcttagatat cggcacaaat agcgtcggat
     1021 gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata
     1081 cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac agtggagaga
     1141 cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt cggaagaatc
     1201 gtatttgtta tctacaggag attttttcaa atgagatggc gaaagtagat gatagtttct
     1261 ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa cgtcatccta
     1321 tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact atctatcatc
     1381 tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc tatttggcct
     1441 tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta aatcctgata
     1501 atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa ttatttgaag
     1561 aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca cgattgagta
     1621 aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa aatggcttat
     1681 ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca aattttgatt
     1741 tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat ttagataatt
     1801 tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag aatttatcag
     1861 atgctatttt actttcagat atcctaagag taaatactga aataactaag gctcccctat
     1921 cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt ttaaaagctt
     1981 tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg
     2041 gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa tttatcaaac
     2101 caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat cgtgaagatt
     2161 tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt cacttgggtg
     2221 agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa gacaatcgtg
     2281 agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca ttggcgcgtg
     2341 gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc ccatggaatt
     2401 ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc atgacaaact
     2461 ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt tatgagtatt
     2521 ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag
     2581 catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa
     2641 aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg
     2701 ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc
     2761 taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg
     2821 atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa
     2881 catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg
     2941 gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa
     3001 caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg cagctgatcc
     3061 atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg
     3121 atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa aaaggtattt
     3181 tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat aagccagaaa
     3241 atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag aaaaattcgc
     3301 gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag attcttaaag
     3361 agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat tatctccaaa
     3421 atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt gattatgatg
     3481 tcgatcacat tgttccacaa agtttcctta aagacgattc aatagacaat aaggtcttaa
     3541 cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa
     3601 agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa cgtaagtttg
     3661 ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct ggttttatca
     3721 aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc
     3781 gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa gtgattacct
     3841 taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa gtacgtgaga
     3901 ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga actgctttga
     3961 ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa gtttatgatg
     4021 ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca aaatatttct
     4081 tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat ggagagattc
     4141 gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg gataaagggc
     4201 gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa
     4261 cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga aattcggaca
     4321 agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt gatagtccaa
     4381 cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg aagaagttaa
     4441 aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt gaaaaaaatc
     4501 cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta atcattaaac
     4561 tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg gctagtgccg
     4621 gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat tttttatatt
     4681 tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa aaacaattgt
     4741 ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt gaattttcta
     4801 agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat aacaaacata
     4861 gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg ttgacgaatc
     4921 ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa cgatatacgt
     4981 ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt ctttatgaaa
     5041 cacgcattga tttgagtcag ctaggaggtg actaaggttg agacctgcag gcttcctcgc
     5101 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5161 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5221 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cacaggctcc
     5281 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5341 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5401 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5461 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5521 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5581 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5641 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5701 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5761 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5821 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5881 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5941 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6001 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6061 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6121 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgcgac ccacgctc
//
