LOCUS       sequence-194488-        7873 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to BDKRB2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7873)
  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 7873)
  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)_P2A-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..7873
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1570..1596
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1612..1635,1636..1638,1639..3213)
                     /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             3244..3300
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3307..3326)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3803..3823
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3940..3960)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4358..4369)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4500..4517)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4506..4713
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4722..4862
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4722..4851
                     /label=AAV2 ITR
     rep_origin      4937..5392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5024..5043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5234..5255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5409..5428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5528..5550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5588..5606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5674..5778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5779..5847,5848..6639)
                     /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(5997..6016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6810..7398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7299..7318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7469..7754
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
       61 ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg
      121 tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg
      181 tgcgccacca tgaagacgat catcgccctg agctacatct tctgcctggt attcgccgac
      241 tacaaggacg atgatgacgc cagcatcgat atgttctcac cctggaagat ttctatgttc
      301 ctttctgtgc gcgaggacag cgtgcccaca actgcgagtt ttagcgccga catgcttaac
      361 gtgaccctgc agggccccac tctcaacggg acatttgctc agtctaaatg tcctcaagtg
      421 gaatggctcg gctggttgaa cactatccag ccccctttcc tttgggttct ttttgttctg
      481 gccacgctgg aaaacatctt tgttctctct gtgttttgcc tccataagag ctcctgtacg
      541 gtggccgaaa tttacctggg taatttggcc gcagcggatc tcattttggc ctgtggcctg
      601 cctttctggg ctattactat cagcaataat tttgattggt tgtttggcga gaccttgtgt
      661 cgcgtggtga atgcaattat cagtatgaat ctgtacagct caatctgctt ccttatgctc
      721 gtcagcattg acaggtacct cgcccttgta aagactatga gcatgggccg catgaggggc
      781 gtacgctggg ctaagctcta tagcctggtg atttggggtt gcaccctcct cctcagttca
      841 cctatgctgg tcttccgaac gatgaaggag tactctgacg agggccacaa tgttaccgca
      901 tgcgttatca gttacccatc tttgatttgg gaggtcttca caaacatgct gctgaacgtg
      961 gtgggttttt tgctccccct ttctgtgatt accttctgta caatgcagat catgcaagtt
     1021 ttgcgaaaca acgaaatgca aaaatttaag gagatacaga ccgagcgaag ggccacagtg
     1081 ctggtcttgg tcgttttgct gttgttcatt atctgttggc tgcctttcca gatcagcact
     1141 ttcctggaca cgttgcacag actcggaatc ctgagtagtt gtcaggacga aaggatcatc
     1201 gacgttatca ctcaaatcgc gtccttcatg gcatactcca atagctgctt gaacccattg
     1261 gtgtatgtta ttgtgggaaa acggtttagg aagaagagct gggaagtgta ccaaggggtg
     1321 tgccaaaagg gaggctgccg atcagagcca atccaaatgg aaaatagcat gggtaccctg
     1381 agaacctcca ttagcgtgga gcgacagatc cacaagctcc aggactgggc tggctctaga
     1441 cagatcgata ccggtggacg caccccaccc agcctgggtc cccaagatga gtcctgcacc
     1501 accgccagct cctccctggc caaggacact tcatcgaccg gtgagaacct gtacttccag
     1561 ctaactagtt acccatacga cgtaccagat tacgctggag gttctggatc cgacaagaag
     1621 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1681 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1741 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1801 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1861 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1921 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1981 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     2041 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     2101 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     2161 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     2221 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     2281 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg
     2341 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     2401 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2461 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2521 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2581 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2641 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2701 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2761 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2821 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2881 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2941 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     3001 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     3061 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     3121 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     3181 accaaagtga aatacgtgac cgagggaatg agaggtggcg gtggctcggg taccggatct
     3241 ggagccacca atttttctct gctgaagcag gctggtgatg ttgaagaaaa ccctggacct
     3301 gaattcatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     3361 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     3421 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     3481 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     3541 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     3601 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     3661 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     3721 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     3781 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     3841 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caaggggtaa
     3901 ctaagtaagg atcgatccaa gatatcgtat tcttaactat gttgctcctt ttacgctatg
     3961 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     4021 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     4081 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     4141 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     4201 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4261 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     4321 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     4381 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4441 gacgagtcgg atctcccttt gggccgcctc cccgccaatt cctagagctc gctgatcagc
     4501 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4561 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4621 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4681 ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga
     4741 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4801 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4861 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4921 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4981 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     5041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     5281 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5401 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5461 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5521 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5581 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5641 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5701 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5761 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5821 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5881 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5941 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6001 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6061 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6121 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6181 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6241 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6301 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6361 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6421 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6481 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6541 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6601 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6661 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6721 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6781 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6841 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6901 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6961 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7021 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7081 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7141 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7201 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7261 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7321 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7381 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7441 tggccttttg ctcacatcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
     7501 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
     7561 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     7621 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     7681 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     7741 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     7801 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     7861 cggggggggg ggg
//
