LOCUS       sequence-239365-        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"
     protein_bind    121..142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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        225..243
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     225..242
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(273..299,300..302,303..2789,2790..2792,2793..4376)
                     /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             4389..4409
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4473..4492)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4474..4492)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4502..4524)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4718..4737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4837..4859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4897..4915)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4983..5087
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5088..5156,5157..5948)
                     /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(5306..5325)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6119..6707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6608..6627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6861..6874,1..4)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacacg agctatttag gtgacactat
      241 agaagtgaag cttggtctca aacagtggat ccatggataa gaagtactct atcggactcg
      301 ctatcggaac taactctgtg ggatgggctg tgatcaccga tgagtacaag gtgccatcta
      361 agaagttcaa ggttctcgga aacaccgata ggcactctat caagaaaaac cttatcggtg
      421 ctctcctctt cgattctggt gaaactgctg aggctaccag actcaagaga accgctagaa
      481 gaaggtacac cagaagaaag aacaggatct gctacctcca agagatcttc tctaacgaga
      541 tggctaaagt ggatgattca ttcttccaca ggctcgaaga gtcattcctc gtggaagaag
      601 ataagaagca cgagaggcac cctatcttcg gaaacatcgt tgatgaggtg gcataccacg
      661 agaagtaccc tactatctac cacctcagaa agaagctcgt tgattctact gataaggctg
      721 atctcaggct catctacctc gctctcgctc acatgatcaa gttcagagga cacttcctca
      781 tcgagggtga tctcaaccct gataactctg atgtggataa gttgttcatc cagctcgtgc
      841 agacctacaa ccagcttttc gaagagaacc ctatcaacgc ttcaggtgtg gatgctaagg
      901 ctatcctctc tgctaggctc tctaagtcaa gaaggcttga gaacctcatt gctcagctcc
      961 ctggtgagaa gaagaacgga cttttcggaa acttgatcgc tctctctctc ggactcaccc
     1021 ctaacttcaa gtctaacttc gatctcgctg aggatgcaaa gctccagctc tcaaaggata
     1081 cctacgatga tgatctcgat aacctcctcg ctcagatcgg agatcagtac gctgatttgt
     1141 tcctcgctgc taagaacctc tctgatgcta tcctcctcag tgatatcctc agagtgaaca
     1201 ccgagatcac caaggctcca ctctcagctt ctatgatcaa gagatacgat gagcaccacc
     1261 aggatctcac acttctcaag gctcttgtta gacagcagct cccagagaag tacaaagaga
     1321 ttttcttcga tcagtctaag aacggatacg ctggttacat cgatggtggt gcatctcaag
     1381 aagagttcta caagttcatc aagcctatcc tcgagaagat ggatggaacc gaggaactcc
     1441 tcgtgaagct caatagagag gatcttctca gaaagcagag gaccttcgat aacggatcta
     1501 tccctcatca gatccacctc ggagagttgc acgctatcct tagaaggcaa gaggatttct
     1561 acccattcct caaggataac agggaaaaga ttgagaagat tctcaccttc agaatccctt
     1621 actacgtggg acctctcgct agaggaaact caagattcgc ttggatgacc agaaagtctg
     1681 aggaaaccat caccccttgg aacttcgaag aggtggtgga taagggtgct agtgctcagt
     1741 ctttcatcga gaggatgacc aacttcgata agaaccttcc aaacgagaag gtgctcccta
     1801 agcactcttt gctctacgag tacttcaccg tgtacaacga gttgaccaag gttaagtacg
     1861 tgaccgaggg aatgaggaag cctgcttttt tgtcaggtga gcaaaagaag gctatcgttg
     1921 atctcttgtt caagaccaac agaaaggtga ccgtgaagca gctcaaagag gattacttca
     1981 agaaaatcga gtgcttcgat tcagttgaga tttctggtgt tgaggatagg ttcaacgcat
     2041 ctctcggaac ctaccacgat ctcctcaaga tcattaagga taaggatttc ttggataacg
     2101 aggaaaacga ggatatcttg gaggatatcg ttcttaccct caccctcttt gaagatagag
     2161 agatgattga agaaaggctc aagacctacg ctcatctctt cgatgataag gtgatgaagc
     2221 agttgaagag aagaagatac actggttggg gaaggctctc aagaaagctc attaacggaa
     2281 tcagggataa gcagtctgga aagacaatcc ttgatttcct caagtctgat ggattcgcta
     2341 acagaaactt catgcagctc atccacgatg attctctcac ctttaaagag gatatccaga
     2401 aggctcaggt ttcaggacag ggtgatagtc tccatgagca tatcgctaac ctcgctggat
     2461 ctcctgcaat caagaaggga atcctccaga ctgtgaaggt tgtggatgag ttggtgaagg
     2521 tgatgggaag gcataagcct gagaacatcg tgatcgaaat ggctagagag aaccagacca
     2581 ctcagaaggg acagaagaac tctagggaaa ggatgaagag gatcgaggaa ggtatcaaag
     2641 agcttggatc tcagatcctc aaagagcacc ctgttgagaa cactcagctc cagaatgaga
     2701 agctctacct ctactacctc cagaacggaa gggatatgta tgtggatcaa gagttggata
     2761 tcaacaggct ctctgattac gatgttgatg ctatcgtgcc acagtcattc ttgaaggatg
     2821 attctatcga taacaaggtg ctcaccaggt ctgataagaa caggggtaag agtgataacg
     2881 tgccaagtga agaggttgtg aagaaaatga agaactattg gaggcagctc ctcaacgcta
     2941 agctcatcac tcagagaaag ttcgataact tgactaaggc tgagagggga ggactctctg
     3001 aattggataa ggcaggattc atcaagaggc agcttgtgga aaccaggcag atcactaagc
     3061 acgttgcaca gatcctcgat tctaggatga acaccaagta cgatgagaac gataagttga
     3121 tcagggaagt gaaggttatc accctcaagt caaagctcgt gtctgatttc agaaaggatt
     3181 tccaattcta caaggtgagg gaaatcaaca actaccacca cgctcacgat gcttacctta
     3241 acgctgttgt tggaaccgct ctcatcaaga agtatcctaa gctcgagtca gagttcgtgt
     3301 acggtgatta caaggtgtac gatgtgagga agatgatcgc taagtctgag caagagatcg
     3361 gaaaggctac cgctaagtat ttcttctact ctaacatcat gaatttcttc aagaccgaga
     3421 ttaccctcgc taacggtgag atcagaaaga ggccactcat cgagacaaac ggtgaaacag
     3481 gtgagatcgt gtgggataag ggaagggatt tcgctaccgt tagaaaggtg ctctctatgc
     3541 cacaggtgaa catcgttaag aaaaccgagg tgcagaccgg tggattctct aaagagtcta
     3601 tcctccctaa gaggaactct gataagctca ttgctaggaa gaaggattgg gaccctaaga
     3661 aatacggtgg tttcgattct cctaccgtgg cttactctgt tctcgttgtg gctaaggttg
     3721 agaagggaaa gagtaagaag ctcaagtctg ttaaggaact tctcggaatc actatcatgg
     3781 aaaggtcatc tttcgagaag aacccaatcg atttcctcga ggctaaggga tacaaagagg
     3841 ttaagaagga tctcatcatc aagctcccaa agtactcact cttcgaactc gagaacggta
     3901 gaaagaggat gctcgcttct gctggtgagc ttcaaaaggg aaacgagctt gctctcccat
     3961 ctaagtacgt taactttctt tacctcgctt ctcactacga gaagttgaag ggatctccag
     4021 aagataacga gcagaagcaa cttttcgttg agcagcacaa gcactacttg gatgagatca
     4081 tcgagcagat ctctgagttc tctaaaaggg tgatcctcgc tgatgcaaac ctcgataagg
     4141 tgttgtctgc ttacaacaag cacagagata agcctatcag ggaacaggca gagaacatca
     4201 tccatctctt cacccttacc aacctcggtg ctcctgctgc tttcaagtac ttcgatacaa
     4261 ccatcgatag gaagagatac acctctacca aagaagtgct cgatgctacc ctcatccatc
     4321 agtctatcac tggactctac gagactagga tcgatctctc acagctcggt ggtgattcaa
     4381 gggctgatcc taagaagaag aggaaggttt gactgcagag gaggtaatca atgccacaac
     4441 actggtggta ccggcttgag accgaattct cgccctatag tgagtcgtat tacctggccg
     4501 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4561 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4621 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     4681 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     4741 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4861 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4921 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4981 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5041 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5101 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5161 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5221 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5281 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5341 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5401 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5461 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5521 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5581 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5641 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5701 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5761 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggctctcga ggtatcattg
     5821 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5881 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5941 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6001 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6061 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6121 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6181 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6241 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6301 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6361 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6421 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6481 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6541 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6601 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6661 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6721 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6781 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6841 gcagccgaac gaccgagcgc agcgagtcag tgag
//
