LOCUS       sequence-197201-        7300 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Encodes dCas9(N)-DHFR fused to BDKRB2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Baeumler TA, Ahmed AA, Fulga TA
  TITLE     Engineering Synthetic Signaling Pathways with Programmable 
            dCas9-Based Chimeric Receptors.
  JOURNAL   Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 
            10.1016/j.celrep.2017.08.044.
  PUBMED    28903044
REFERENCE   2  (bases 1 to 7300)
  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"; doi: 
            "10.1016/j.celrep.2017.08"; journalName: "Cell Rep"; date: 
            "2017-09-12- 12"; volume: "20"; issue: "11"; pages: "2639-2653"
COMMENT     Sequence Label: pBDKRB2_TCS(Q'L)_HA-dCas9(N)_DHFR
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          110..337
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1737..1763
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1779..1802,1803..1805,1806..3380)
                     /codon_start=1
                     /product="N-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the D10A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             3408..3887
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     primer_bind     complement(3929..3946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3935..4142
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4151..4291
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4151..4280
                     /label=AAV2 ITR
     rep_origin      4366..4821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4453..4472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4663..4684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4838..4857)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4957..4979
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5017..5035)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5103..5207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5208..5276,5277..6068)
                     /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(5426..5445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6239..6827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6728..6747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6898..7183
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
       61 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      121 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      181 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      241 ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa
      301 ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc gccaccatga
      361 agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac aaggacgatg
      421 atgacgccag catcgatatg ttctcaccct ggaagatttc tatgttcctt tctgtgcgcg
      481 aggacagcgt gcccacaact gcgagtttta gcgccgacat gcttaacgtg accctgcagg
      541 gccccactct caacgggaca tttgctcagt ctaaatgtcc tcaagtggaa tggctcggct
      601 ggttgaacac tatccagccc cctttccttt gggttctttt tgttctggcc acgctggaaa
      661 acatctttgt tctctctgtg ttttgcctcc ataagagctc ctgtacggtg gccgaaattt
      721 acctgggtaa tttggccgca gcggatctca ttttggcctg tggcctgcct ttctgggcta
      781 ttactatcag caataatttt gattggttgt ttggcgagac cttgtgtcgc gtggtgaatg
      841 caattatcag tatgaatctg tacagctcaa tctgcttcct tatgctcgtc agcattgaca
      901 ggtacctcgc ccttgtaaag actatgagca tgggccgcat gaggggcgta cgctgggcta
      961 agctctatag cctggtgatt tggggttgca ccctcctcct cagttcacct atgctggtct
     1021 tccgaacgat gaaggagtac tctgacgagg gccacaatgt taccgcatgc gttatcagtt
     1081 acccatcttt gatttgggag gtcttcacaa acatgctgct gaacgtggtg ggttttttgc
     1141 tccccctttc tgtgattacc ttctgtacaa tgcagatcat gcaagttttg cgaaacaacg
     1201 aaatgcaaaa atttaaggag atacagaccg agcgaagggc cacagtgctg gtcttggtcg
     1261 ttttgctgtt gttcattatc tgttggctgc ctttccagat cagcactttc ctggacacgt
     1321 tgcacagact cggaatcctg agtagttgtc aggacgaaag gatcatcgac gttatcactc
     1381 aaatcgcgtc cttcatggca tactccaata gctgcttgaa cccattggtg tatgttattg
     1441 tgggaaaacg gtttaggaag aagagctggg aagtgtacca aggggtgtgc caaaagggag
     1501 gctgccgatc agagccaatc caaatggaaa atagcatggg taccctgaga acctccatta
     1561 gcgtggagcg acagatccac aagctccagg actgggctgg ctctagacag atcgataccg
     1621 gtggacgcac cccacccagc ctgggtcccc aagatgagtc ctgcaccacc gccagctcct
     1681 ccctggccaa ggacacttca tcgaccggtg agaacctgta cttccagcta actagttacc
     1741 catacgacgt accagattac gctggaggtt ctggatccga caagaagtac agcatcggcc
     1801 tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     1861 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     1921 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     1981 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     2041 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     2101 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     2161 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     2221 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     2281 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     2341 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     2401 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     2461 tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga
     2521 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     2581 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     2641 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     2701 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     2761 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     2821 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     2881 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     2941 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     3001 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     3061 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     3121 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     3181 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     3241 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     3301 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     3361 acgtgaccga gggaatgaga ggtggcggtg gctcgggtac cggatctatg atcagtctga
     3421 ttgcggcgtt agcggtagat tacgttatcg gcatggaaaa cgccatgccg tggaacctgc
     3481 ctgccgatct cgcctggttt aaacgcaaca ccttaaataa acccgtgatt atgggccgcc
     3541 atacctggga atcaatcggt cgtccgttgc caggacgcaa aaatattatc ctcagcagtc
     3601 aaccgagtac ggacgatcgc gtaacgtggg tgaagtcggt ggatgaagcc atcgcggcgt
     3661 gtggtgacgt accagaaatc atggtgattg gcggcggtcg cgttattgaa cagttcttgc
     3721 caaaagcgca aaaactgtat ctgacgcata tcgacgcaga agtggaaggc gacacccatt
     3781 tcccggatta cgagccggat gactgggaat cggtattcag cgaattccac gatgctgatg
     3841 cgcagaactc tcacagctat tgctttgaga ttctggagcg gcgataaaag cttcgcctcc
     3901 ccgccaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3961 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4021 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4081 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     4141 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4201 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4261 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4321 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4381 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4441 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4501 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4561 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4621 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4681 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4741 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4801 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4861 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4921 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4981 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5041 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5101 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5161 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5221 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5281 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5341 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5401 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5461 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5521 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5581 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5641 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5701 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5761 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5821 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5881 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     5941 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6001 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6061 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6121 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6181 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6241 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6301 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6361 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6421 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6481 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6541 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6601 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6661 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6721 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6781 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6841 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatctag aggtacccgt
     6901 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     6961 gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     7021 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     7081 caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc
     7141 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     7201 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     7261 tttttaatta ttttgtgcag cgatgggggc gggggggggg
//
