LOCUS       sequence-236028-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo golden gate assembly CD part for dCas9 (Catalytically-inactive
            S. pyogenes Cas9, for gRNA-targeted repression). Please see 
            Supplemental Documents for annotated Genbank file..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6211)
  TITLE     CIDAR MoClo Extension
REFERENCE   2  (bases 1 to 6211)
  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: C41m
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..71
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(75..118)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    121..142
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(158..184,185..187,188..2674,2675..2677,2678..4264)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_feature    4275..4295
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(4301..4318)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4472..5060)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4552..4571)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5231..6022,6023..6091))
                     /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     5854..5873
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6092..6196)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
       61 tatcacgagg cagaatttca gataaaaaaa atccttagct ttcgctaagg atgatttctg
      121 gaattcgcgg ccgcttctag agactagtgg gtctcaaatg gataagaaat actcaatagg
      181 cttagctatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc
      241 gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat
      301 aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc
      361 tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa
      421 tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga
      481 agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta
      541 tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa
      601 agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt
      661 tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt
      721 ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg gagtagatgc
      781 taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc tcattgctca
      841 gctccccggt gagaagaaaa atggcttatt tgggaatctc attgctttgt cattgggttt
      901 gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac agctttcaaa
      961 agatacttac gatgatgatt tagataattt attggcgcaa attggagatc aatatgctga
     1021 tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata tcctaagagt
     1081 aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct acgatgaaca
     1141 tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag aaaagtataa
     1201 agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg ggggagctag
     1261 ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg gtactgagga
     1321 attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct ttgacaacgg
     1381 ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa gacaagaaga
     1441 cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga cttttcgaat
     1501 tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa
     1561 gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag gtgcttcagc
     1621 tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg aaaaagtact
     1681 accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa
     1741 atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat
     1801 tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta
     1861 tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa
     1921 tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga
     1981 taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga
     2041 tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat
     2101 gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa
     2161 tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt
     2221 tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat
     2281 tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc
     2341 tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt
     2401 caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca
     2461 gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat
     2521 caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa
     2581 tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt
     2641 agatattaat cgtttaagtg attatgatgt cgatgccatt gttccacaaa gtttccttaa
     2701 agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga
     2761 taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa
     2821 cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt
     2881 gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac
     2941 taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa
     3001 acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa
     3061 agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta
     3121 tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt
     3181 tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga
     3241 aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac
     3301 agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga
     3361 aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc
     3421 catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga
     3481 gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc
     3541 aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa
     3601 ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat
     3661 tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa
     3721 ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa
     3781 cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct
     3841 gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag
     3901 tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga
     3961 gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga
     4021 taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa
     4081 tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga
     4141 tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat
     4201 ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga
     4261 ctaaggtaga gacctactag tagcggccgc tgcaggcttc ctcgctcact gactcgctgc
     4321 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4381 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4441 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag gctccgcccc cctgacgagc
     4501 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4561 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4621 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4681 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4741 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4801 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4861 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4921 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4981 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5041 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5101 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5161 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5221 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5281 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5341 catctggccc cagtgctgca atgataccgc gcgacccacg ctcaccggct ccagatttat
     5401 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5461 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5521 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5581 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5641 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5701 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5761 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5821 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5881 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5941 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6001 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6061 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6121 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6181 aaataggggt tccgcgcaca tttccccgaa a
//
