LOCUS       sequence-236320-        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"
     CDS             complement(join(80..871,872..940))
                     /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"
     primer_bind     703..722
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(941..1045)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1113..1131
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1135..1178)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             1201..5307
                     /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             1201..5304
                     /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(5328..5345)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5499..6087)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5579..5598)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
       61 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
      121 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
      181 agggcttacc atctggcccc agtgctgcaa tgataccgcg cgacccacgc tcaccggctc
      241 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
      301 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
      361 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
      421 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
      481 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
      541 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
      601 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
      661 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
      721 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
      781 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
      841 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
      901 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
      961 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1021 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     1081 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaatttca
     1141 gataaaaaaa atccttagct ttcgctaagg atgatttctg gaattcgcgg ccggtctcaa
     1201 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg
     1261 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
     1321 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
     1381 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
     1441 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
     1501 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
     1561 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
     1621 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
     1681 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
     1741 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
     1801 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
     1861 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
     1921 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
     1981 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
     2041 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
     2101 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
     2161 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
     2221 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
     2281 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
     2341 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
     2401 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
     2461 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
     2521 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
     2581 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
     2641 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
     2701 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
     2761 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
     2821 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
     2881 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
     2941 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
     3001 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
     3061 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
     3121 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
     3181 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
     3241 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
     3301 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
     3361 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
     3421 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
     3481 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
     3541 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
     3601 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga
     3661 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac
     3721 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
     3781 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
     3841 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
     3901 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
     3961 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
     4021 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
     4081 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
     4141 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
     4201 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
     4261 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
     4321 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc
     4381 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
     4441 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
     4501 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
     4561 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
     4621 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
     4681 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
     4741 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
     4801 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
     4861 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
     4921 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
     4981 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
     5041 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
     5101 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
     5161 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
     5221 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
     5281 gatttgagtc agctaggagg tgactaaggt tgagacctgc aggcttcctc gctcactgac
     5341 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5401 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5461 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccacaggct ccgcccccct
     5521 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5581 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5641 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5701 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5761 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5821 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5881 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5941 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6001 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6061 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6121 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaagga
//
