LOCUS       sequence-405405-        8671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9-RPT6 with FLAG label.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8671)
  AUTHORS   Farrell K, Auerbach A, Musaus M, Navabpour S, Liu C, Lin Y, Xie H, 
            Jarome TJ
  TITLE     Phosphorylation of RPT6 Controls Its Ability to Bind DNA and 
            Regulate Gene Expression in the Hippocampus of Male Rats during 
            Memory Formation.
  JOURNAL   J Neurosci. 2024 Jan 24;44(4):e1453232023. doi: 
            10.1523/JNEUROSCI.1453-23.2023.
  PUBMED    38124005
REFERENCE   2  (bases 1 to 8671)
  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: "J 
            Neurosci."; date: "2024-01-24"; volume: "44(4):e1453232023. doi"; 
            pages: " 10.1523/JNEUROSCI.1453-23.2023"
COMMENT     Sequence Label: dCas9-RPT6-FLAG
FEATURES             Location/Qualifiers
     source          1..8671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(748..774,775..777,778..3261,3262..3267,3268..3333,
                     3334..3336,3337..4851)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6175..6195
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6205..6270
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(6325..6344)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6369..6417
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(6731..7319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6811..6830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7490..8281,8282..8350))
                     /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     8113..8132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8351..8455)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8530..8549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc
      781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
      961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1261 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3241 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct
     3301 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc
     3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4861 gaccccaaga agaagaggaa ggtgagtggt ggaggaagtg gcgggtcagg gtcgatggac
     4921 gcgaaatcac ttacggcatg gtcggcgctt gatgggccag agcagatgga actggaagag
     4981 gggaaggcag gcagtggact ccgtcaatat tatctgtcca agattgaaga actccagttg
     5041 attgtgaatg acaagagtca gaacctccgg agactgcagg cccagaggaa tgagctcaat
     5101 gcaaaagttc gtctgttgcg ggaggaactg cagctgctgc aggaacaggg ctcctacgtt
     5161 ggagaagtcg tgagggccat ggataagaaa aaagtgttgg tcaaggtcca tcctgagggc
     5221 aaatttgttg ttgatgtgga caagaacatt gatatcaatg atgtgacacc caattgtcgg
     5281 gtggctctaa gaaatgacag ctacactctg cataagatcc tgcctaacaa agtggaccct
     5341 ttggtatcac taatgatggt ggaaaaggtg ccagactcaa cctacgagat gattggcggg
     5401 ctagacaagc agatcaagga gatcaaagaa gtgattgagc tgcctgtgaa gcaccctgag
     5461 ctctttgaag ccctgggcat tgcacagcca aagggagtcc tgctatatgg acccccgggc
     5521 actgggaaga cgttgttggc ccgggctgtg gctcatcata cggactgtac ctttattcgt
     5581 gtctctggct ctgaactggt acagaaattc attggggaag gggcaagaat ggtgagggag
     5641 ctgtttgtca tggcccgaga acatgctccc tccatcatct tcatggacga gatcgactct
     5701 atcggttctt cacggctgga gggaggctct ggaggcgaca gtgaggtaca gcgcacaatg
     5761 ctggaactgc tcaaccagct ggatggcttt gaggccacta agaatatcaa ggttatcatg
     5821 gctactaata ggattgatat cctggactct gccctgcttc gtcctgggag gattgacagg
     5881 aaaattgaat tcccacctcc caatgaggag gcccggctgg acattttgaa aatccactct
     5941 cggaaaatga acttgaccag ggggatcaac ctgaggaaaa ttgctgagtt gatgccagga
     6001 gcttcagggg ctgaagtgaa gggcgtgtgc acagaggccg gcatgtatgc tctgcgggaa
     6061 cggcgtgtcc acgttactca ggaggacttt gagatggcag tagccaaggt catgcaaaag
     6121 gatagtgaga aaaacatgtc catcaagaag ctttggaagg gctctggatc aagcccaaag
     6181 aagaaacgga aggtggaagc ctcagactat aaggaccacg acggagacta caaggatcat
     6241 gatattgatt acaaagacga tgacgataag tgaaagaatt cgatccctac cggttagtaa
     6301 tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     6361 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     6421 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     6481 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     6541 cagggctcgc agccaacgtc ggggcggcag gccctccata gtcggtcgtt cggctgcggc
     6601 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6661 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6721 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6781 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6841 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6901 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6961 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7021 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7081 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7141 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7201 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7261 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7321 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7381 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7441 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7501 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7561 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7621 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7681 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7741 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7801 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7861 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7921 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7981 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8041 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8101 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8161 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8221 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8281 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8341 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8401 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8461 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     8521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     8581 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     8641 gaccgacaat tgcatgaaga atctgcttag g
//
