LOCUS       sequence-197202-        7353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dCas9(N)-DHFR fused to BDKRB2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7353)
  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 7353)
  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..7353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2204..2230
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2246..2269,2270..2272,2273..3847)
                     /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             3875..4354
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     primer_bind     complement(4396..4413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4402..4609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4618..4758
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4618..4747
                     /label=AAV2 ITR
     rep_origin      4833..5288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4920..4939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5130..5151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5305..5324)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5424..5446
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5484..5502)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5570..5674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5675..5743,5744..6535)
                     /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(5893..5912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6706..7294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7195..7214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggtgcgcc accatgaaga cgatcatcgc
      841 cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat
      901 cgatatgttc tcaccctgga agatttctat gttcctttct gtgcgcgagg acagcgtgcc
      961 cacaactgcg agttttagcg ccgacatgct taacgtgacc ctgcagggcc ccactctcaa
     1021 cgggacattt gctcagtcta aatgtcctca agtggaatgg ctcggctggt tgaacactat
     1081 ccagccccct ttcctttggg ttctttttgt tctggccacg ctggaaaaca tctttgttct
     1141 ctctgtgttt tgcctccata agagctcctg tacggtggcc gaaatttacc tgggtaattt
     1201 ggccgcagcg gatctcattt tggcctgtgg cctgcctttc tgggctatta ctatcagcaa
     1261 taattttgat tggttgtttg gcgagacctt gtgtcgcgtg gtgaatgcaa ttatcagtat
     1321 gaatctgtac agctcaatct gcttccttat gctcgtcagc attgacaggt acctcgccct
     1381 tgtaaagact atgagcatgg gccgcatgag gggcgtacgc tgggctaagc tctatagcct
     1441 ggtgatttgg ggttgcaccc tcctcctcag ttcacctatg ctggtcttcc gaacgatgaa
     1501 ggagtactct gacgagggcc acaatgttac cgcatgcgtt atcagttacc catctttgat
     1561 ttgggaggtc ttcacaaaca tgctgctgaa cgtggtgggt tttttgctcc ccctttctgt
     1621 gattaccttc tgtacaatgc agatcatgca agttttgcga aacaacgaaa tgcaaaaatt
     1681 taaggagata cagaccgagc gaagggccac agtgctggtc ttggtcgttt tgctgttgtt
     1741 cattatctgt tggctgcctt tccagatcag cactttcctg gacacgttgc acagactcgg
     1801 aatcctgagt agttgtcagg acgaaaggat catcgacgtt atcactcaaa tcgcgtcctt
     1861 catggcatac tccaatagct gcttgaaccc attggtgtat gttattgtgg gaaaacggtt
     1921 taggaagaag agctgggaag tgtaccaagg ggtgtgccaa aagggaggct gccgatcaga
     1981 gccaatccaa atggaaaata gcatgggtac cctgagaacc tccattagcg tggagcgaca
     2041 gatccacaag ctccaggact gggctggctc tagacagatc gataccggtg gacgcacccc
     2101 acccagcctg ggtccccaag atgagtcctg caccaccgcc agctcctccc tggccaagga
     2161 cacttcatcg accggtgaga acctgtactt ccagctaact agttacccat acgacgtacc
     2221 agattacgct ggaggttctg gatccgacaa gaagtacagc atcggcctgg ccatcggcac
     2281 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     2341 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     2401 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     2461 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     2521 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     2581 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     2641 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     2701 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     2761 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     2821 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     2881 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     2941 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     3001 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     3061 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     3121 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     3181 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     3241 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     3301 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     3361 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     3421 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     3481 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     3541 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     3601 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     3661 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     3721 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     3781 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     3841 aatgagaggt ggcggtggct cgggtaccgg atctatgatc agtctgattg cggcgttagc
     3901 ggtagattac gttatcggca tggaaaacgc catgccgtgg aacctgcctg ccgatctcgc
     3961 ctggtttaaa cgcaacacct taaataaacc cgtgattatg ggccgccata cctgggaatc
     4021 aatcggtcgt ccgttgccag gacgcaaaaa tattatcctc agcagtcaac cgagtacgga
     4081 cgatcgcgta acgtgggtga agtcggtgga tgaagccatc gcggcgtgtg gtgacgtacc
     4141 agaaatcatg gtgattggcg gcggtcgcgt tattgaacag ttcttgccaa aagcgcaaaa
     4201 actgtatctg acgcatatcg acgcagaagt ggaaggcgac acccatttcc cggattacga
     4261 gccggatgac tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactctca
     4321 cagctattgc tttgagattc tggagcggcg ataaaagctt cgcctccccg ccaattccta
     4381 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4441 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4501 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4561 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     4621 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4681 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4741 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4801 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5041 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5101 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5161 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5221 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5281 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5341 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5401 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5461 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5521 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5581 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5641 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5701 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5761 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5821 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5881 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5941 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6001 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6061 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6121 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6181 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6241 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6301 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6361 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     6421 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6481 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6541 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6601 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6661 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6721 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6781 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6841 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6901 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6961 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7021 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7081 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7141 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7201 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7261 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7321 gttcctggcc ttttgctggc cttttgctca cat
//
