LOCUS       sequence-231125-        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     467..486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(705..809)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     877..895
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(899..942)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    945..966
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(982..1008,1009..1011,1012..3498,3499..3501,3502..5088)
                     /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    5099..5119
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(5125..5142)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5296..5884)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5376..5395)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6055..6211,1..635,636..704))
                     /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 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
       61 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
      121 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
      181 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
      241 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
      301 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
      361 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
      421 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
      481 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
      541 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
      601 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
      661 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
      721 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
      781 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
      841 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcagaat
      901 ttcagataaa aaaaatcctt agctttcgct aaggatgatt tctggaattc gcggccgctt
      961 ctagagacta gtgggtctca aatggataag aaatactcaa taggcttagc tatcggcaca
     1021 aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag
     1081 gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt
     1141 gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca
     1201 cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta
     1261 gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga taagaagcat
     1321 gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca
     1381 actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta
     1441 atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat
     1501 ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat
     1561 caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct
     1621 gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag
     1681 aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa
     1741 tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat
     1801 gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct
     1861 aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact
     1921 aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact
     1981 cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat
     2041 caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat
     2101 aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta
     2161 aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa
     2221 attcacttgg gtgagctgca tgctattttg agaaggcaag aggactttta tccattttta
     2281 aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt
     2341 ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt
     2401 accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa
     2461 cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg
     2521 ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga
     2581 atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc
     2641 aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa
     2701 tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc
     2761 taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa
     2821 gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag
     2881 gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt
     2941 cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag
     3001 caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt
     3061 atgcagctga tccatgatga tagtttgaca tttaaagaag atattcaaaa agcacaagtg
     3121 tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt
     3181 aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg
     3241 cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc
     3301 cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt
     3361 cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc
     3421 tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta
     3481 agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga ttcaatagac
     3541 aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa
     3601 gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact
     3661 caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa
     3721 gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa
     3781 attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt
     3841 aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat
     3901 aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt
     3961 ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat
     4021 aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc
     4081 gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca
     4141 aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc
     4201 tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat
     4261 attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa
     4321 agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt
     4381 tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa
     4441 tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc
     4501 tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac
     4561 ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg
     4621 ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg
     4681 aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa
     4741 caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc
     4801 agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca
     4861 tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt
     4921 acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt
     4981 aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact
     5041 ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactaagg tagagaccta
     5101 ctagtagcgg ccgctgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5161 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5221 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5281 cgcgttgctg gcgtttttcc acaggctccg cccccctgac gagcatcaca aaaatcgacg
     5341 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5401 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5461 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5521 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5581 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5641 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5701 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5761 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5821 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5881 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5941 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6001 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6061 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6121 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6181 tgcaatgata ccgcgcgacc cacgctcacc g
//
