LOCUS       sequence-426182-        6764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning A.thaliana codon optimized SpCas9 CDS as GreenGate module B 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6764)
  AUTHORS   Dona M, Bradamante G, Bogojevic Z, Gutzat R, Streubel S, Mosiolek M,
            Dolan L, Mittelsten Scheid O
  TITLE     A versatile CRISPR-based system for lineage tracing in living 
            plants.
  JOURNAL   Plant J. 2023 Jul 5. doi: 10.1111/tpj.16378.
  PUBMED    37403571
REFERENCE   2  (bases 1 to 6764)
  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"; journalName: "Plant J. 
            2023 Jul 5. doi: 10.1111/tpj.16378."
COMMENT     Sequence Label: pGGB-SpCas9
FEATURES             Location/Qualifiers
     source          1..6764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             92..4195
                     /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             92..4195
                     /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             4208..4228
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(4299..4317)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4300..4317)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4861..4878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5032..5620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5112..5131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5791..6582,6583..6651))
                     /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     6414..6433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6652..6755)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taatacgact cactataggg agacgcacat gcggccgcct cgagaattct gacgtcttaa
       61 ttaattatta tcagggtggt ggtctccaac aatggataag aagtactcta tcggactcga
      121 tatcggaact aactctgtgg gatgggctgt gatcaccgat gagtacaagg tgccatctaa
      181 gaagttcaag gttctcggaa acaccgatag gcactctatc aagaaaaacc ttatcggtgc
      241 tctcctcttc gattctggtg aaactgctga ggctaccaga ctcaagagaa ccgctagaag
      301 aaggtacacc agaagaaaga acaggatctg ctacctccaa gagatcttct ctaacgagat
      361 ggctaaagtg gatgattcat tcttccacag gctcgaagag tcattcctcg tggaagaaga
      421 taagaagcac gagaggcacc ctatcttcgg aaacatcgtt gatgaggtgg cataccacga
      481 gaagtaccct actatctacc acctcagaaa gaagctcgtt gattctactg ataaggctga
      541 tctcaggctc atctacctcg ctctcgctca catgatcaag ttcagaggac acttcctcat
      601 cgagggtgat ctcaaccctg ataactctga tgtggataag ttgttcatcc agctcgtgca
      661 gacctacaac cagcttttcg aagagaaccc tatcaacgct tcaggtgtgg atgctaaggc
      721 tatcctctct gctaggctct ctaagtcaag aaggcttgag aacctcattg ctcagctccc
      781 tggtgagaag aagaacggac ttttcggaaa cttgatcgct ctctctctcg gactcacccc
      841 taacttcaag tctaacttcg atctcgctga ggatgcaaag ctccagctct caaaggatac
      901 ctacgatgat gatctcgata acctcctcgc tcagatcgga gatcagtacg ctgatttgtt
      961 cctcgctgct aagaacctct ctgatgctat cctcctcagt gatatcctca gagtgaacac
     1021 cgagatcacc aaggctccac tctcagcttc tatgatcaag agatacgatg agcaccacca
     1081 ggatctcaca cttctcaagg ctcttgttag acagcagctc ccagagaagt acaaagagat
     1141 tttcttcgat cagtctaaga acggatacgc tggttacatc gatggtggtg catctcaaga
     1201 agagttctac aagttcatca agcctatcct cgagaagatg gatggaaccg aggaactcct
     1261 cgtgaagctc aatagagagg atcttctcag aaagcagagg accttcgata acggatctat
     1321 ccctcatcag atccacctcg gagagttgca cgctatcctt agaaggcaag aggatttcta
     1381 cccattcctc aaggataaca gggaaaagat tgagaagatt ctcaccttca gaatccctta
     1441 ctacgtggga cctctcgcta gaggaaactc aagattcgct tggatgacca gaaagtctga
     1501 ggaaaccatc accccttgga acttcgaaga ggtggtggat aagggtgcta gtgctcagtc
     1561 tttcatcgag aggatgacca acttcgataa gaaccttcca aacgagaagg tgctccctaa
     1621 gcactctttg ctctacgagt acttcaccgt gtacaacgag ttgaccaagg ttaagtacgt
     1681 gaccgaggga atgaggaagc ctgctttttt gtcaggtgag caaaagaagg ctatcgttga
     1741 tctcttgttc aagaccaaca gaaaggtgac cgtgaagcag ctcaaagagg attacttcaa
     1801 gaaaatcgag tgcttcgatt cagttgagat ttctggtgtt gaggataggt tcaacgcatc
     1861 tctcggaacc taccacgatc tcctcaagat cattaaggat aaggatttct tggataacga
     1921 ggaaaacgag gatatcttgg aggatatcgt tcttaccctc accctctttg aagatagaga
     1981 gatgattgaa gaaaggctca agacctacgc tcatctcttc gatgataagg tgatgaagca
     2041 gttgaagaga agaagataca ctggttgggg aaggctctca agaaagctca ttaacggaat
     2101 cagggataag cagtctggaa agacaatcct tgatttcctc aagtctgatg gattcgctaa
     2161 cagaaacttc atgcagctca tccacgatga ttctctcacc tttaaagagg atatccagaa
     2221 ggctcaggtt tcaggacagg gtgatagtct ccatgagcat atcgctaacc tcgctggatc
     2281 tcctgcaatc aagaagggaa tcctccagac tgtgaaggtt gtggatgagt tggtgaaggt
     2341 gatgggaagg cataagcctg agaacatcgt gatcgaaatg gctagagaga accagaccac
     2401 tcagaaggga cagaagaact ctagggaaag gatgaagagg atcgaggaag gtatcaaaga
     2461 gcttggatct cagatcctca aagagcaccc tgttgagaac actcagctcc agaatgagaa
     2521 gctctacctc tactacctcc agaacggaag ggatatgtat gtggatcaag agttggatat
     2581 caacaggctc tctgattacg atgttgatca tatcgtgcca cagtcattct tgaaggatga
     2641 ttctatcgat aacaaggtgc tcaccaggtc tgataagaac aggggtaaga gtgataacgt
     2701 gccaagtgaa gaggttgtga agaaaatgaa gaactattgg aggcagctcc tcaacgctaa
     2761 gctcatcact cagagaaagt tcgataactt gactaaggct gagaggggag gactctctga
     2821 attggataag gcaggattca tcaagaggca gcttgtggaa accaggcaga tcactaagca
     2881 cgttgcacag atcctcgatt ctaggatgaa caccaagtac gatgagaacg ataagttgat
     2941 cagggaagtg aaggttatca ccctcaagtc aaagctcgtg tctgatttca gaaaggattt
     3001 ccaattctac aaggtgaggg aaatcaacaa ctaccaccac gctcacgatg cttaccttaa
     3061 cgctgttgtt ggaaccgctc tcatcaagaa gtatcctaag ctcgagtcag agttcgtgta
     3121 cggtgattac aaggtgtacg atgtgaggaa gatgatcgct aagtctgagc aagagatcgg
     3181 aaaggctacc gctaagtatt tcttctactc taacatcatg aatttcttca agaccgagat
     3241 taccctcgct aacggtgaga tcagaaagag gccactcatc gagacaaacg gtgaaacagg
     3301 tgagatcgtg tgggataagg gaagggattt cgctaccgtt agaaaggtgc tctctatgcc
     3361 acaggtgaac atcgttaaga aaaccgaggt gcagaccggt ggattctcta aagagtctat
     3421 cctccctaag aggaactctg ataagctcat tgctaggaag aaggattggg accctaagaa
     3481 atacggtggt ttcgattctc ctaccgtggc ttactctgtt ctcgttgtgg ctaaggttga
     3541 gaagggaaag agtaagaagc tcaagtctgt taaggaactt ctcggaatca ctatcatgga
     3601 aaggtcatct ttcgagaaga acccaatcga tttcctcgag gctaagggat acaaagaggt
     3661 taagaaggat ctcatcatca agctcccaaa gtactcactc ttcgaactcg agaacggtag
     3721 aaagaggatg ctcgcttctg ctggtgagct tcaaaaggga aacgagcttg ctctcccatc
     3781 taagtacgtt aactttcttt acctcgcttc tcactacgag aagttgaagg gatctccaga
     3841 agataacgag cagaagcaac ttttcgttga gcagcacaag cactacttgg atgagatcat
     3901 cgagcagatc tctgagttct ctaaaagggt gatcctcgct gatgcaaacc tcgataaggt
     3961 gttgtctgct tacaacaagc acagagataa gcctatcagg gaacaggcag agaacatcat
     4021 ccatctcttc acccttacca acctcggtgc tcctgctgct ttcaagtact tcgatacaac
     4081 catcgatagg aagagataca cctctaccaa agaagtgctc gatgctaccc tcatccatca
     4141 gtctatcact ggactctacg agactaggat cgatctctca cagctcggtg gtgattcaag
     4201 ggctgatcct aagaagaaga ggaaggtttg aggggctcga gaccggtggt atcatggttt
     4261 ttacctcctg aattcggatc cctcgagcga tacacacttc tatagtgtca cctaaatgcg
     4321 tttaaacctt cctgcaggtg acgattacct aacaatcggt cgattcgttt gatgttatgt
     4381 tttgttctcg ctttggttgg caggttacgg ccaagttcgg taagagtgag agttttacag
     4441 tcaagtaatg cgtggcaagc caacgttaag ctgttgagtc gttttaagtg taattcgggg
     4501 cagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat
     4561 tgatttttcg cgaaaccatt taatcatatg acaagatgtg tgtccacctt aacttaatga
     4621 tttttaccaa aatcattagg ggattcatca gcgctgagtg tgtaaattaa tttttatgcc
     4681 gcagcgggcc agcaattctc gtgaatcatc gcttaaacgg cctgatttct gaatagctat
     4741 gcgtaagcgg gtgttgtcta acattctgcg ttcctcttta tcctgtctga accggctgca
     4801 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4861 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4921 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4981 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgca ttgctggcgt ttttccatag
     5041 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5101 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5161 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5221 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5281 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5401 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5461 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5521 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5581 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5641 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5701 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5761 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5821 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5881 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     5941 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6001 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6061 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6121 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6181 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6241 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6301 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6361 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6421 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6481 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6541 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6601 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6661 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6721 atgtatttag aaaaataaac aaataggggt tccgcccgcg aaat
//
