LOCUS       sequence-245424-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo golden gate assembly CD part for Cas9 (S. pyogenes 
            gRNA-targeted endonuclease Cas9, with bbsI cut sites removed 
            synonymously). 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: C114m
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..44
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(48..91)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    94..115
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(131..157,158..160,161..2647,2648..2650,2651..4237)
                     /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    4248..4268
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(4274..4291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4445..5033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4525..4544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5204..5995,5996..6064))
                     /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     5827..5846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6065..6169)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattatcatg acattaacct ataaaaatag gcgtatcacg aggcagaatt tcagataaaa
       61 aaaatcctta gctttcgcta aggatgattt ctggaattcg cggccgcttc tagagactag
      121 tgggtctcaa atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg
      181 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
      241 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
      301 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
      361 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
      421 ctttcatcga cttgaagagt cttttttggt ggaagaagat aagaagcatg aacgtcatcc
      481 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
      541 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
      601 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
      661 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
      721 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
      781 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
      841 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
      901 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
      961 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
     1021 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     1081 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     1141 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     1201 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     1261 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     1321 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     1381 tgagctgcat gctattttga gaaggcaaga ggacttttat ccatttttaa aagacaatcg
     1441 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
     1501 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     1561 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     1621 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     1681 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     1741 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     1801 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     1861 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     1921 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     1981 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     2041 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     2101 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     2161 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     2221 ccatgatgat agtttgacat ttaaagaaga tattcaaaaa gcacaagtgt ctggacaagg
     2281 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     2341 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     2401 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     2461 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     2521 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     2581 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     2641 tgtcgatgcc attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     2701 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     2761 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     2821 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     2881 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     2941 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     3001 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     3061 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     3121 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     3181 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     3241 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     3301 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     3361 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
     3421 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
     3481 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
     3541 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
     3601 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
     3661 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
     3721 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
     3781 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
     3841 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
     3901 gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc
     3961 taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca
     4021 tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa
     4081 tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac
     4141 gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga
     4201 aacacgcatt gatttgagtc agctaggagg tgactaaggt agagacctac tagtagcggc
     4261 cgctgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4321 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4381 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4441 cgtttttcca caggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4501 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4561 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4621 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4681 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4741 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4801 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4861 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4921 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4981 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5041 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5101 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5161 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5221 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5281 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5341 cgcgcgaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5401 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5461 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5521 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5581 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5641 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5701 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5761 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5821 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5881 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5941 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6001 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6061 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6121 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6181 gaaaagtgcc acctgacgtc taagaaacca t
//
