LOCUS       sequence-234541-        6874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with GreenGate B-C flanking sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6874)
  AUTHORS   Lee JE, Neumann M, Duro DI, Schmid M
  TITLE     CRISPR-based tools for targeted transcriptional and epigenetic 
            regulation in plants.
  JOURNAL   PLoS One. 2019 Sep 26;14(9):e0222778. doi: 
            10.1371/journal.pone.0222778. eCollection 2019.
  PUBMED    31557222
REFERENCE   2  (bases 1 to 6874)
  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: "PLoS One.";
            date: "2019-09-26"; volume: "14(9):e0222778. doi"; pages: " 
            10.1371/journal.pone.0222778. eCollection 2019"
COMMENT     Sequence Label: pMH220
FEATURES             Location/Qualifiers
     source          1..6874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        2212..2230
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2212..2229
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(2260..2286,2287..2289,2290..4776,4777..4779,
                     4780..6363)
                     /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"
     CDS             6376..6396
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6460..6479)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6461..6479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6489..6511)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6705..6724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6824..6846
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtggctct cgaggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacgagc tatttaggtg
     2221 acactataga agtgaagctt ggtctcaaac agtggatcca tggataagaa gtactctatc
     2281 ggactcgcta tcggaactaa ctctgtggga tgggctgtga tcaccgatga gtacaaggtg
     2341 ccatctaaga agttcaaggt tctcggaaac accgataggc actctatcaa gaaaaacctt
     2401 atcggtgctc tcctcttcga ttctggtgaa actgctgagg ctaccagact caagagaacc
     2461 gctagaagaa ggtacaccag aagaaagaac aggatctgct acctccaaga gatcttctct
     2521 aacgagatgg ctaaagtgga tgattcattc ttccacaggc tcgaagagtc attcctcgtg
     2581 gaagaagata agaagcacga gaggcaccct atcttcggaa acatcgttga tgaggtggca
     2641 taccacgaga agtaccctac tatctaccac ctcagaaaga agctcgttga ttctactgat
     2701 aaggctgatc tcaggctcat ctacctcgct ctcgctcaca tgatcaagtt cagaggacac
     2761 ttcctcatcg agggtgatct caaccctgat aactctgatg tggataagtt gttcatccag
     2821 ctcgtgcaga cctacaacca gcttttcgaa gagaacccta tcaacgcttc aggtgtggat
     2881 gctaaggcta tcctctctgc taggctctct aagtcaagaa ggcttgagaa cctcattgct
     2941 cagctccctg gtgagaagaa gaacggactt ttcggaaact tgatcgctct ctctctcgga
     3001 ctcaccccta acttcaagtc taacttcgat ctcgctgagg atgcaaagct ccagctctca
     3061 aaggatacct acgatgatga tctcgataac ctcctcgctc agatcggaga tcagtacgct
     3121 gatttgttcc tcgctgctaa gaacctctct gatgctatcc tcctcagtga tatcctcaga
     3181 gtgaacaccg agatcaccaa ggctccactc tcagcttcta tgatcaagag atacgatgag
     3241 caccaccagg atctcacact tctcaaggct cttgttagac agcagctccc agagaagtac
     3301 aaagagattt tcttcgatca gtctaagaac ggatacgctg gttacatcga tggtggtgca
     3361 tctcaagaag agttctacaa gttcatcaag cctatcctcg agaagatgga tggaaccgag
     3421 gaactcctcg tgaagctcaa tagagaggat cttctcagaa agcagaggac cttcgataac
     3481 ggatctatcc ctcatcagat ccacctcgga gagttgcacg ctatccttag aaggcaagag
     3541 gatttctacc cattcctcaa ggataacagg gaaaagattg agaagattct caccttcaga
     3601 atcccttact acgtgggacc tctcgctaga ggaaactcaa gattcgcttg gatgaccaga
     3661 aagtctgagg aaaccatcac cccttggaac ttcgaagagg tggtggataa gggtgctagt
     3721 gctcagtctt tcatcgagag gatgaccaac ttcgataaga accttccaaa cgagaaggtg
     3781 ctccctaagc actctttgct ctacgagtac ttcaccgtgt acaacgagtt gaccaaggtt
     3841 aagtacgtga ccgagggaat gaggaagcct gcttttttgt caggtgagca aaagaaggct
     3901 atcgttgatc tcttgttcaa gaccaacaga aaggtgaccg tgaagcagct caaagaggat
     3961 tacttcaaga aaatcgagtg cttcgattca gttgagattt ctggtgttga ggataggttc
     4021 aacgcatctc tcggaaccta ccacgatctc ctcaagatca ttaaggataa ggatttcttg
     4081 gataacgagg aaaacgagga tatcttggag gatatcgttc ttaccctcac cctctttgaa
     4141 gatagagaga tgattgaaga aaggctcaag acctacgctc atctcttcga tgataaggtg
     4201 atgaagcagt tgaagagaag aagatacact ggttggggaa ggctctcaag aaagctcatt
     4261 aacggaatca gggataagca gtctggaaag acaatccttg atttcctcaa gtctgatgga
     4321 ttcgctaaca gaaacttcat gcagctcatc cacgatgatt ctctcacctt taaagaggat
     4381 atccagaagg ctcaggtttc aggacagggt gatagtctcc atgagcatat cgctaacctc
     4441 gctggatctc ctgcaatcaa gaagggaatc ctccagactg tgaaggttgt ggatgagttg
     4501 gtgaaggtga tgggaaggca taagcctgag aacatcgtga tcgaaatggc tagagagaac
     4561 cagaccactc agaagggaca gaagaactct agggaaagga tgaagaggat cgaggaaggt
     4621 atcaaagagc ttggatctca gatcctcaaa gagcaccctg ttgagaacac tcagctccag
     4681 aatgagaagc tctacctcta ctacctccag aacggaaggg atatgtatgt ggatcaagag
     4741 ttggatatca acaggctctc tgattacgat gttgatgcta tcgtgccaca gtcattcttg
     4801 aaggatgatt ctatcgataa caaggtgctc accaggtctg ataagaacag gggtaagagt
     4861 gataacgtgc caagtgaaga ggttgtgaag aaaatgaaga actattggag gcagctcctc
     4921 aacgctaagc tcatcactca gagaaagttc gataacttga ctaaggctga gaggggagga
     4981 ctctctgaat tggataaggc aggattcatc aagaggcagc ttgtggaaac caggcagatc
     5041 actaagcacg ttgcacagat cctcgattct aggatgaaca ccaagtacga tgagaacgat
     5101 aagttgatca gggaagtgaa ggttatcacc ctcaagtcaa agctcgtgtc tgatttcaga
     5161 aaggatttcc aattctacaa ggtgagggaa atcaacaact accaccacgc tcacgatgct
     5221 taccttaacg ctgttgttgg aaccgctctc atcaagaagt atcctaagct cgagtcagag
     5281 ttcgtgtacg gtgattacaa ggtgtacgat gtgaggaaga tgatcgctaa gtctgagcaa
     5341 gagatcggaa aggctaccgc taagtatttc ttctactcta acatcatgaa tttcttcaag
     5401 accgagatta ccctcgctaa cggtgagatc agaaagaggc cactcatcga gacaaacggt
     5461 gaaacaggtg agatcgtgtg ggataaggga agggatttcg ctaccgttag aaaggtgctc
     5521 tctatgccac aggtgaacat cgttaagaaa accgaggtgc agaccggtgg attctctaaa
     5581 gagtctatcc tccctaagag gaactctgat aagctcattg ctaggaagaa ggattgggac
     5641 cctaagaaat acggtggttt cgattctcct accgtggctt actctgttct cgttgtggct
     5701 aaggttgaga agggaaagag taagaagctc aagtctgtta aggaacttct cggaatcact
     5761 atcatggaaa ggtcatcttt cgagaagaac ccaatcgatt tcctcgaggc taagggatac
     5821 aaagaggtta agaaggatct catcatcaag ctcccaaagt actcactctt cgaactcgag
     5881 aacggtagaa agaggatgct cgcttctgct ggtgagcttc aaaagggaaa cgagcttgct
     5941 ctcccatcta agtacgttaa ctttctttac ctcgcttctc actacgagaa gttgaaggga
     6001 tctccagaag ataacgagca gaagcaactt ttcgttgagc agcacaagca ctacttggat
     6061 gagatcatcg agcagatctc tgagttctct aaaagggtga tcctcgctga tgcaaacctc
     6121 gataaggtgt tgtctgctta caacaagcac agagataagc ctatcaggga acaggcagag
     6181 aacatcatcc atctcttcac ccttaccaac ctcggtgctc ctgctgcttt caagtacttc
     6241 gatacaacca tcgataggaa gagatacacc tctaccaaag aagtgctcga tgctaccctc
     6301 atccatcagt ctatcactgg actctacgag actaggatcg atctctcaca gctcggtggt
     6361 gattcaaggg ctgatcctaa gaagaagagg aaggtttgac tgcagaggag gtaatcaatg
     6421 ccacaacact ggtggtaccg gcttgagacc gaattctcgc cctatagtga gtcgtattac
     6481 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     6541 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     6601 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     6661 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     6721 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6781 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6841 cagaggtttt caccgtcatc accgaaacgc gcga
//
